Methods of using crystals of beta amyloid cleaving enzyme (BACE)

ABSTRACT

Methods of using monoclinic crystals of human beta-secretase (BACE) having unit cell dimensions of a, b, and c, wherein a is about 81±20 Å to about 101 Å, b is 103±20 Å, c is 100±20 Å, and α=γ=90°, and β is 105°±10° for crystals of symmetry P2 1  and a=73.1, b=105.1, c=50.5 Å and β is 94.8° for crystals of C2 symmetry in drug screening assays comprising selecting a potential modifier by adding the potential modifiers to an aqueous mixture of the crystal and detecting a measure of binding, such that the potential modifier that binds is selected as a potential drug.

RELATED APPLICATIONS

This application is a divisional application and claims the benefit ofU.S. application Ser. No. 10/143,723, filed May 10, 2002, now U.S. Pat.No. 7,524,668, which claims the benefit of the U.S. ProvisionalApplication Ser. No. 60/290,120, filed May 10, 2001, and 60/334,648,filed Nov. 30, 2001, both of which are incorporated herein by referencein their entireties.

This application incorporates by reference the material contained on theduplicate (2) compact discs submitted herewith. Each disc contains thefollowing files.

Name Size Contents Date of File Creation table_1.txt 735 kbytes Table 1Nov. 15, 2001 table_2.txt 273 kbytes Table 2 Nov. 15, 2001 table_3.txt470 kbytes Table 3 May 9, 2001

FIELD OF THE INVENTION

This invention relates to the crystallization and structuredetermination of BACE, also known as memapsin 2 and beta secretase, fromhuman (Homo sapiens), particularly in a form as produced in an E. coliexpression system.

BACKGROUND

Alzheimer's disease (AD) causes progressive dementia with consequentformation of amyloid plaques, neurofibrillary tangles, gliosis andneuronal loss. The disease occurs in both genetic and sporadic formswhose clinical course and pathological features are quite similar. Threegenes have been discovered to date which, when mutated, cause anautosomal dominant form of Alzheimer's disease. These encode the amyloidprotein precursor (APP) and two related proteins, presenilin-1 (PS1) andpresenilin-2 (PS2), which, as their names suggest, are structurally andfunctionally related. Mutations in any of the three proteins have beenobserved to enhance proteolytic processing of APP via an intracellularpathway that produces amyloid beta peptide. (Aβ peptide, or sometimeshere as Abeta), a 40-42 amino acid long peptide that is the primarycomponent of amyloid plaque in AD.

Dysregulation of intracellular pathways for proteolytic processing maybe central to the pathophysiology of AD. In the case of plaqueformation, mutations in APP, PS1 or PS2 consistently alter theproteolytic processing of APP so as to enhance formation of Aβ 1-42, aform of the Aβ peptide which seems to be particularly amyloidogenic, andthus very important in AD. Different forms of APP range in size from695-770 amino acids, localize to the cell surface, and have a singleC-terminal transmembrane domain. The Abeta peptide is derived from aregion of APP adjacent to and containing a portion of the transmembranedomain. Normally, processing of APP at the α-secretase site cleaves themidregion of the Aβ sequence adjacent to the membrane and releases thesoluble, extracellular domain of APP from the cell surface. Thisα-secretase APP processing creates soluble APP-α, which is normal andnot thought to contribute to AD. Pathological processing of APP at theβ- and γ-secretase sites, which are located N-terminal and C-terminal tothe α-secretase site, respectively, produces a very different resultthan processing at the α site. Sequential processing at the β- andγ-secretase sites releases the Aβ peptide, a peptide possibly veryimportant in AD pathogenesis. Processing at the β- and γ-secretase sitescan occur in both the endoplasmic reticulum (in neurons) and in theendosomal/lysosomal pathway after reinternalization of cell surface APP(in all cells). Despite intense efforts, for 10 years or more, toidentify the enzymes responsible for processing APP at the β and γsites, to produce the Aβ peptide, those proteases remained unknown untilrecently. The identification and characterization of the β secretaseenzyme, termed Aspartyl Protease 2 (Asp2) or BACE has been established.Since the β-secretase catalyzes the committed step in formation of theAβ peptide, it has become a key target in the search for therapeuticagents to combat Alzheimer's disease. It is believed that inhibition ofBACE should slow or stop the onset of amyloid plaque formation and theassociated symptoms of Alzheimer's disease.

In addition, the X-ray crystal structure of human BACE in complex with apeptide inhibitor was solved and published (Hong et al., Science290:150-53 (2000)) from protein expressed in E. coli that contained nocovalent sugar (glycosylation) at any of the four putative glycosylationsites within the enzyme.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a crystal of BACEincluding a unit cell defined by the dimensions a, b, c, α, β, and γ,wherein a is about 61 Å to about 101 Å, b is about 83 Å to about 123 Å,c is about 80 Å to about 120 Å, α=γ=90°, and β is about 95° to about115°. Preferably, the crystal has monoclinic space group symmetry P2₁.

In another aspect, the present invention provides a crystal of BACEhaving monoclinic space group symmetry C2.

In another aspect, the present invention provides a crystal of BACEincluding a unit cell having dimensions of a, b, and c, wherein a isabout 53 Å to about 103 Å, b is about 85 Å to about 125 Å, and c isabout 40 Å to about 60 Å; and α=γ=90°, and β is about 85° to about 105°.Preferably, the crystal has monoclinic space group symmetry C2.

The present invention also provides methods of using the crystals ofBACE in a drug screening assay.

In another aspect, the present invention provides a method forcrystallizing a human BACE molecule or molecular complex. In oneembodiment, the method includes preparing purified human BACE in thepresence of a potential modifier, and crystallizing the human BACE froma solution including the purified BACE and the potential modifier,wherein the solution has a pH of about 4.5 to about 5.6. In anotherembodiment, the method includes preparing purified human BACE in thepresence of a potential modifier, and adding a precipitant salt to asolution including the purified BACE and the potential modifier.

In another aspect, the present invention provides a molecule ormolecular complex that that forms a crystal having a unit cell definedby the dimensions a, b, c, α, β, and γ, wherein a is about 61 Å to about101 Å, b is about 83 Å to about 123 Å, c is about 80 Å to about 120 Å,α=γ=90°, and β is about 95° to about 115°, and that includes at least aportion of a human BACE or BACE-like binding pocket, wherein the bindingpocket includes the amino acids listed in Table 4A, the binding pocketbeing defined by a set of points having a root mean square deviation ofless than about 0.65 Å from points representing the backbone atoms ofsaid amino acids as represented by the structure coordinates listed inTable 1.

In another aspect, the present invention provides a molecule ormolecular complex that forms a crystal having monoclinic space groupsymmetry C2, and that includes at least a portion of a human BACEbinding pocket, wherein the binding pocket includes the amino acidslisted in Table 4B, the binding pocket being defined by a set of pointshaving a root mean square deviation of less than about 0.65 Å frompoints representing the backbone atoms of said amino acids asrepresented by the structure coordinates listed in Table 2.

In another aspect, the present invention provides a scalablethree-dimensional configuration of points, at least a portion of saidpoints being derived from structure coordinates of at least a portion ofa human BACE molecule or molecular complex listed in Table 1 including ahuman BACE or BACE-like binding pocket, wherein the human BACE moleculeor molecular complex forms a crystal having a unit cell defined by thedimensions a, b, c, α, β, and γ, wherein a is about 61 Å to about 101 Å,b is about 83 Å to about 123 Å, c is about 80 Å to about 120 Å, α=γ=90°,and β is about 95° to about 115°. Preferably, the scalablethree-dimensional configuration of points is displayed as a holographicimage, a stereodiagram, a model, or a computer-displayed image.

In another aspect, the present invention provides a scalablethree-dimensional configuration of points, at least a portion of saidpoints being derived from structure coordinates of at least a portion ofa human BACE molecule or molecular complex listed in Table 2 including ahuman BACE or BACE-like binding pocket, wherein the human BACE moleculeor molecular complex forms a crystal having monoclinic space groupsymmetry C2.

In another aspect, the present invention provides a machine-readabledata storage medium including a data storage material encoded withmachine readable data which, when using a machine programmed withinstructions for using said data, displays a graphical three-dimensionalrepresentation of a molecule or molecular complex. In one embodiment themolecule or molecular complex includes at least a portion of a humanBACE or BACE-like binding pocket including the amino acids listed inTable 4A, the binding pocket defined by a set of points having a rootmean square deviation of less than about 0.65 Å from points representingthe backbone atoms of said amino acids as represented by structurecoordinates listed in Table 1 of a human BACE or BACE-like molecule ormolecular complex that forms a crystal having a unit cell defined by thedimensions a, b, c, α, β, and γ, wherein a is about 61 Å to about 101 Å,b is about 83 Å about 123 Å, c is about 80 Å to about 120 Å, α=γ=90°,and β is about 95° to about 115°. In another embodiment, the molecule ormolecular complex includes at least a portion of a human BACE orBACE-like binding pocket including the amino acids listed in Table 4B,the binding pocket defined by a set of points having a root mean squaredeviation of less than about 0.65 Å from points representing thebackbone atoms of said amino acids as represented by structurecoordinates listed in Table 2 of a human BACE or BACE-like molecule ormolecular complex that forms a crystal having monoclinic space groupsymmetry C2.

In another aspect, the present invention provides a method for obtainingstructural information about a molecule or a molecular complex ofunknown structure. In one embodiment, the method includes crystallizingthe molecule or molecular complex; generating an x-ray diffractionpattern from the crystallized molecule or molecular complex; andapplying to the x-ray diffraction pattern at least a portion of thestructure coordinates as set forth in Table 1 for human BACE that formsa crystal having a unit cell defined by the dimensions a, b, c, α, β,and γ, wherein a is about 61 Å to about 101 Å, b is about 83 Å to about123 Å, c is about 80 Å to about 120 Å, α=γ=90°, and β is about 95° toabout 115°, to generate a three-dimensional electron density map of atleast a portion of the molecule or molecular complex whose structure isunknown. In another embodiment, the method includes crystallizing themolecule or molecular complex; generating an x-ray diffraction patternfrom the crystallized molecule or molecular complex; and applying atleast a portion of the structure coordinates for human BACE set forth inTable 2 for human BACE that forms a crystal having monoclinic spacegroup symmetry C2 to the x-ray diffraction pattern to generate athree-dimensional electron density map of at least a portion of themolecule or molecular complex whose structure is unknown.

In another aspect, the present invention provides a method for homologymodeling a human BACE homolog. In one embodiment, the method includesaligning the amino acid sequence of a human BACE homolog with an aminoacid sequence of human BACE and incorporating the sequence of the humanBACE homolog into a model of human BACE formed from structurecoordinates as set forth in Table 1 for human BACE that forms a crystalhaving a unit cell defined by the dimensions a, b, c, α, β, and γ,wherein a is about 61 Å to about 101 Å, b is about 83 Å to about 123 Å,c is about 80 Å to about 120 Å, α=γ90°, and β is about 95° to about115°, to yield a preliminary model of the human BACE homolog; subjectingthe preliminary model to energy minimization to yield an energyminimized model; and remodeling regions of the energy minimized modelwhere stereochemistry restraints are violated to yield a final model ofthe human BACE homolog. In another embodiment, the method includesaligning the amino acid sequence of a human BACE homolog with an aminoacid sequence of human BACE and incorporating the sequence of the humanBACE homolog into a model of human BACE derived from human BACEstructure coordinates set forth in Table 2 for human BACE that forms acrystal having monoclinic space group symmetry C2 to yield a preliminarymodel of the human BACE homolog; subjecting the preliminary model toenergy minimization to yield an energy minimized model; and remodelingregions of the energy minimized model where stereochemistry restraintsare violated to yield a final model of the human BACE homolog.

In another aspect, the present invention provides computer-assistedmethods for identifying, designing, or making a potential modifier ofhuman beta secretase activity. Preferably the methods include screeninga library of chemical entities.

ABBREVIATIONS

The following abbreviations may be used throughout this disclosure:

-   Alzheimer's disease (AD)-   3-[(1,1-Dimethylhydroxyethyl)amino]-2-hydroxy-1-propanesulfonic acid    (AMPSO)-   Amyloid beta peptide (AO peptide or Abeta)-   Amyloid protein precursor (APP)-   Aspartyl protease 2 (Asp2)-   Beta amyloid cleaving enzyme (BACE, memapsin 2, beta secretase)-   β-Mercaptoethanol (BME)-   3-Cyclohexylamino-1-propanesulfonic acid (CAPS)-   Dimethyl sulfoxide (DMSO)-   Ethylenediaminetetraacetic acid (EDTA)-   4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-   2-Methyl-2,4-pentanediol (MPD)-   4-Morpholineetbanesulfonic acid (MES)-   Multiple anomalous dispersion (MAD)-   Presenilin-1 (PS1)-   Presenilin-2 (PS2)-   Poly(ethylene glycol) (PEG)-   2-Amino-2-hydroxymethyl-1,3-propanediol (TRIS)-   TE-TRIS-EDTA

The following amino acid abbreviations are used throughout thisdisclosure:

A = Ala = Alanine T = Thr = Threonine V = Val = Valine C = Cys =Cysteine L = Leu = Leucine Y = Tyr = Tyrosine I = Ile = Isoleucine N =Asn = Asparagine P = Pro = Proline Q = Gln = Glutamine F = Phe =Phenylalanine D = Asp = Aspartic Acid W = Trp = Tryptophan E = Glu =Glutamic Acid M = Met = Methionine K = Lys = Lysine G = Gly = Glycine R= Arg = Arginine S = Ser = Serine H = His = Histidine

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an illustration of the chemical structure of an inhibitor usedin co-crystallization experiments.

FIG. 2 is the synthetic peptideSer-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Arg-Gly-Gly-Cys (where Sta=statine)(SEQ ID NO:4) used for affinity purification of BACE.

FIG. 3 is an Fo-Fc electron density map for the inhibitor illustrated inFIG. 1 at 2.15 Å contoured at 2σ for the P2₁ crystal form.

FIG. 4 is an Fo-Fc electron density map for the inhibitor illustrated inFIG. 1 at 1.7 Å contoured at 2σ for the C2 crystal form.

FIG. 5 is a stereo view of a C^(α) trace of three monomers in theasymmetric unit of the P2₁ crystal form of human BACE (grey line) in thepresence of the inhibitor illustrated in FIG. 1. The inhibitor trace isindicated by a dark black line.

FIG. 6 is a stereo view of a C^(α) trace of one monomer in theasymmetric unit of the C2 crystal form of human BACE (grey line) in thepresence of the inhibitor illustrated in FIG. 1. The general location ofthe inhibitor trace (black line) is indicated by an arrow.

FIG. 7 depicts a stereo view of the active site of human BACE from theP2₁ crystal form (illustrated with light gray carbons, dark grayoxygens, and black nitrogens) with the inhibitor illustrated in FIG. 1.The general location of the inhibitor is indicated by an arrow(illustrated with light gray carbons, dark gray oxygens, and blacknitrogens). The iodine atom is circled and the two fluorine atoms areindicated by a “•” symbol.

FIG. 8 depicts a stereo view of the active site of human BACE from theC2 crystal form (illustrated with light gray carbons, dark gray oxygens,and black nitrogens) with the inhibitor illustrated in FIG. 1. Thegeneral location of the inhibitor is indicated by an arrow (illustratedwith light gray carbons, dark gray oxygens, and black nitrogens). Theiodine atom is circled and the two fluorine atoms are indicated by a “•”symbol.

FIG. 9 depicts distance of the interactions between the inhibitor andthe active site of BACE. Amino acid residues or main chain atoms within3.5 Å of the inhibitor are shown. Van der Waals interactions are shownwith arcs.

FIG. 10 depicts the sequences of the three E. coli constructs forrecombinant human BACE (SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3) usedto obtain the crystals described. The first visible residue in thecrystal structures is indicated.

FIG. 11 depicts the sequence alignment of the E. coli expressedrecombinant human beta sectetase (SEQ ID NO:1) and the CHO cellexpressed recombinant human beta secretase proteolytically cleaved withHIV protease (SEQ ID NO:5) present in the crystal structure.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Tables 1, 2, and 3 list atomic structure coordinates derived by x-raydiffraction of crystals having space groups P2₁, C2, and P4₃2₁2,respectively, of human BACE expressed in E. coli. Column 2 lists anumber for the atom in the structure. Column 3 lists the element whosecoordinates are measured. The first letter in the column defines theelement. Column 4 lists the type of amino acid. Column 5 lists a numberfor the amino acid in the structure. Columns 6-8 list thecrystallographic coordinates X, Y, and Z respectively. Thecrystallographic coordinates define the atomic position of the elementmeasured. Column 9 lists an occupancy factor that refers to the fractionof the molecules in which each atom occupies the position specified bythe coordinates. A value of “1” indicates that each atom has the sameconformation, i.e., the same position, in all molecules of the crystal.Column 10 lists a thermal factor “B” that measures movement of the atomaround its atomic center. Column 11 lists the chain id (AA for moleculeA in the asymmetric unit, BB for molecule B in the asymmetric unit, CCfor molecule C in the asymmetric unit, WW for water molecules, and LLfor inhibitor molecules). Column 12 lists the element whose coordinatesare measured.

Crystalline Form(s) and Method of Making

The three-dimensional structure of human BACE was solved using x-raycrystallography to 2.15 Å resolution for the P2₁ crystal form and 1.7 Åresolution for the C2 crystal form. Accordingly, the invention includesa human BACE crystal and/or a crystal with human BACE co-crystallizedwith a ligand. As used herein, “ligand” refers to a chemical entity thatcan form a reversible complex with the protein and that could functionas a drug candidate (e.g., modifiers and inhibitors). Thus, the term“ligand” as used herein does not include chemical entities that couldnot function as a drug candidate (e.g., water, metal ions, andsolvents). The crystal has monoclinic space group symmetry P2₁ or C2.The crystal includes monoclinic shaped unit cells, the P2₁ unit cellpreferably having dimensions in which a=81±20 Å, b=103±20 Å, c=100±20 Å,α=γ=90°, β=105±10°, and the C2 unit cell preferably having dimensions inwhich a=73±20 Å, b=105±20 Å, c=50±10 Å, α=γ=90°, β=95±10°. The P2₁crystal form is a monomer with three monomers in the asymmetric unit andthe C2 crystal form is a monomer with a single monomer in the asymmetricunit.

According to the present invention, human BACE can be isolated from avariety of bacterial expression systems, for example, the E. coli strainBL21.

As used herein, a “molecular complex” means a protein in covalent ornon-covalent association with a chemical entity (e.g., a ligand). In oneembodiment, molecular complexes of purified human BACE at aconcentration of about 1 mg/ml to about 80 mg/ml in a solution of about100 mM sodium borate (e.g., pH 8.5) may be crystallized in the presenceof a modifier at a concentration of about 0.001 to about 10 mM.Optionally, the solution includes about 0% by volume to about 40% byvolume organic solvent (e.g., DMSO). Preferably, the solution isbuffered to a pH of about 4.0 to about 6.5 and more preferably about 4.5to about 5.6.

In another embodiment, molecular complexes of purified human BACE at aconcentration of about 1 mg/ml to about 80 mg/ml in a solution of 100 mMsodium borate pH 8.5 may be crystallized in the presence of an inhibitorat a concentration from about 0.001 to about 10 mM, for example, from asolution including about 4% by weight/volume (w/v) to about 50% by w/vof PEG (e.g., PEG 3000) as the precipitant, and about 0% by volume toabout 40% by volume organic solvent (such as DMSO), wherein the solutionis buffered to a pH of about 4.0 to about 6.5 (preferably, a pH of about4.5 to about 5.6). In addition, the percent weight/volume of theprecipitant, e.g., PEG, may be greater than 30% when utilizing lowermolecular weight PEG. For example, the crystallization procedure mayinclude between about 4% weight/volume to about 40% weight/volume of PEG750.

Optionally, the solution may include, for example, at most about 40% byweight ethylene glycol or glycerol. The solution may optionally includeat most about 40% by weight of an organic solvent (e.g.,dimethylsulfoxide or 2-methyl-2,4-pentanediol).

A buffer having a pK_(a) of about 3 to about 7.5 is preferred for use inthe crystallization method. A particularly preferred buffer is about 10mM to about 200 mM ammonium citrate. Variation in buffer and buffer pH,phosphate salts as well as other additives such as PEG, PEG-MME,PEG-DME, or polyoxyalkylenepolyamines is apparent to those skilled inthe art and may result in similar crystals.

Optionally, crystallization of the molecular complex may be induced bythe addition of a precipitant salt to the solution. Precipitant saltsfor precipitating proteins are well known to those of skill in the art,and candidate precipitant salts can easily be screened for their abilityto precipitate the desired protein. Precipitant salts include, forexample, sodium hydrogenphosphate, sodium dihydrogenphosphate, potassiumhydrogenphosphate, potassium dihydrogenphosphate, ammonium phosphate,ammonium sulfate, potassium phosphate, sodium citrate, sodium malonate,sodium acetate, sodium tartrate, ammonium formate, magnesium sulfate,and combinations thereof. A preferred precipitant salt includes, forexample, ammonium phosphate. When precipitant salt is added to thesolution, the solution preferably includes about 0.001 M to about 2.5 Msalt.

The invention further includes a human BACE crystal that is isomorphouswith a human BACE crystal having a unit cell defined by the dimensionsa, b, c, α, β, and γ, wherein a is about 61 Å to about 101 Å, b is about83 Å to about 123 Å, c is about 80 Å to about 120 Å; α=γ=90°, and β isabout 95° to about 115°; or wherein a is about 53 Å to about 93 Å, b isabout 85 Å to about 125 Å, and c is about 40 Å to about 60 Å; andα=γ=90°, and β is about 85° to about 125°.

X-Ray Crystallographic Analysis

Each of the constituent amino acids of human BACE is defined by a set ofstructure coordinates as set forth in Table 1 or Table 2. The term“structure coordinates” refers to Cartesian coordinates derived frommathematical equations related to the patterns obtained on diffractionof a monochromatic beam of x-rays by the atoms (scattering centers) of ahuman BACE complex in crystal form. The diffraction data are used tocalculate an electron density map of the repeating unit of the crystal.The electron density maps are then used to establish the positions ofthe individual atoms of the human BACE protein or protein/ligandcomplex.

Slight variations in structure coordinates can be generated bymathematically manipulating the human BACE or human BACE/ligandstructure coordinates. For example, the structure coordinates set forthin Table 1 or Table 2 could be manipulated by crystallographicpermutations of the structure coordinates, fractionalization of thestructure coordinates, integer additions or subtractions to sets of thestructure coordinates, inversion of the structure coordinates or anycombination of the above. Alternatively, modifications in the crystalstructure due to mutations, additions, substitutions, and/or deletionsof amino acids, or other changes in any of the components that make upthe crystal, could also yield variations in structure coordinates. Suchslight variations in the individual coordinates will have little effecton overall shape. If such variations are within an acceptable standarderror as compared to the original coordinates, the resultingthree-dimensional shape is considered to be structurally equivalent.Structural equivalence is described in more detail below.

It should be noted that slight variations in individual structurecoordinates of the human BACE would not be expected to significantlyalter the nature of chemical entities such as ligands that couldassociate with the binding pockets. In this context, the phrase“associating with” refers to a condition of proximity between a chemicalentity, or portions thereof, and a human BACE molecule or portionsthereof. The association may be non-covalent, wherein the juxtapositionis energetically favored by hydrogen bonding, van der Waals forces, orelectrostatic interactions, or it may be covalent.

Thus, for example, a ligand that bound to a binding pocket of human BACEwould also be expected to bind to or interfere with a structurallyequivalent binding pocket.

For the purpose of this invention, any molecule or molecular complex orbinding pocket thereof, or any portion thereof, that has a root meansquare deviation of conserved residue backbone atoms (N, Cα, C, O) ofless than about 0.65 Å, when superimposed on the relevant backbone atomsdescribed by the reference structure coordinates listed in Table 1and/or Table 2, is considered “structurally equivalent” to the referencemolecule. That is to say, the crystal structures of those portions ofthe two molecules are substantially identical, within acceptable error.As used herein, “residue” refers to one or more atoms. Particularlypreferred structurally equivalent molecules or molecular complexes arethose that are defined by the entire set of structure coordinates listedin Table 1 or Table 2± a root mean square deviation from the conservedbackbone atoms of those amino acids of less than about 0.65 Å. Morepreferably, the root mean square deviation is at most about 0.5 Å. Otherembodiments of this invention include a molecular complex defined by thestructure coordinates listed in Table 1 for those amino acids listed inTable 4A, Table 5A, or Table 6A, ± a root mean square deviation from theconserved backbone atoms of those amino acids of less than about 0.65 Å,preferably at most about 0.5 Å. Still another embodiment of thisinvention includes a molecular complex defined by the structurecoordinates listed in Table 2 for those amino acids listed in Table 4B,Table 5B, or Table 6B, ± a root mean square deviation from the conservedbackbone atoms of those amino acids of less than about 0.65 Å,preferably at most about 0.5 Å.

The term “root mean square deviation” means the square root of thearithmetic mean of the squares of the deviations. It is a way to expressthe deviation or variation from a trend or object. For purposes of thisinvention, the “root mean square deviation” defines the variation in thebackbone of a protein from the backbone of human BACE or a bindingpocket portion thereof, as defined by the structure coordinates of humanBACE described herein.

It will be readily apparent to those of skill in the art that thenumbering of amino acids in other isoforms of human BACE may bedifferent than that of human BACE expressed in E. coli.

Active Site and Other Structural Features

Applicants' invention provides information about the shape and structureof the binding pocket of human BACE in the presence of a modifier. Thesecondary structure of the human BACE monomer includes two domainsconsistent with a typical aspartic protease fold.

Binding pockets are of significant utility in fields such as drugdiscovery. The association of natural ligands or substrates with thebinding pockets of their corresponding receptors or enzymes is the basisof many biological mechanisms of action. Similarly, many drugs exerttheir biological effects through association with the binding pockets ofreceptors and enzymes. Such associations may occur with all or any partsof the binding pocket. An understanding of such associations helps leadto the design of drugs having more favorable associations with theirtarget, and thus improved biological effects. Therefore, thisinformation is valuable in designing potential modifiers of BACE-likebinding pockets, as discussed in more detail below.

The term “binding pocket,” as used herein, refers to a region of amolecule or molecular complex, that, as a result of its shape, favorablyassociates with another chemical entity. Thus, a binding pocket mayinclude or consist of features such as cavities, surfaces, or interfacesbetween domains. Chemical entities that may associate with a bindingpocket include, but are not limited to, cofactors, substrates,inhibitors, agonists, and antagonists.

The amino acid constituents of a human BACE binding pocket as definedherein are positioned in three dimensions in accordance with thestructure coordinates listed in Table 1 or Table 2. In one aspect, thestructure coordinates defining a binding pocket of human BACE includestructure coordinates of all atoms in the constituent amino acids; inanother aspect, the structure coordinates of a binding pocket includestructure coordinates of just the backbone atoms of the constituentamino acids.

The binding pocket of human BACE may include the amino acids listed inTable 4A, more preferably the amino acids listed in Table 5A, and mostpreferably the amino acids listed in Table 6A, as represented by thestructure coordinates listed in Table 1. The binding pocket of humanBACE may include the amino acids listed in Table 4B, more preferably theamino acids listed in Table 5B, and most preferably the amino acidslisted in Table 6B, as represented by the structure coordinates listedin Table 2. Alternatively, the binding pocket of human BACE may bedefined by those amino acids whose backbone atoms are situated withinabout 4 Å, more preferably within about 7 Å, most preferably withinabout 10 Å, of one or more constituent atoms of a bound substrate ormodifier. In yet another alternative, the binding pocket may be definedby those amino acids whose backbone atoms are situated within a spherecentered on the coordinates representing the alpha carbon atom ofresidue Thr 231, the sphere having a radius of about 15 Å, preferablyabout 20 Å, and more preferably about 25 Å.

The term “BACE-like binding pocket” refers to a portion of a molecule ormolecular complex whose shape is sufficiently similar to at least aportion of a binding pocket of human BACE as to be expected to bindrelated structural analogues. As used herein, “at least a portion” meansthat at least about 50% of the amino acids are included, preferably atleast about 70% of the amino acids are included, more preferably atleast about 90% of the amino acids are included, and most preferably allthe amino acids are included. A structurally equivalent binding pocketis defined by a root mean square deviation from the structurecoordinates of the backbone atoms of the amino acids that make upbinding pockets in human BACE (as set forth in Table 1 or Table 2) of atmost about 0.35 Å. How this calculation is obtained is described below.

Accordingly, the invention provides molecules or molecular complexesincluding a human BACE binding pocket or BACE-like binding pocket, asdefined by the sets of structure coordinates described above.

TABLE 4A Residues with 4 Å of the binding site for the P2₁ crystal form.GLY 11 GLY 13 LEU 30 ASP 32 GLY 34 SER 35 PRO 70 TYR 71 THR 72 GLN 73GLY 74 PHE 108 ILE 110 TRP 115 TYR 198 ILE 226 ASP 228 GLY 230 THR 231THR 232 ARG 235

TABLE 4B Residues within 4 Å of the binding site for the C2 crystalform. GLY 11 GLY 12 GLY 13 LEU 30 ASP 32 GLY 34 SER 35 PRO 70 TYR 71 THR72 GLN 73 GLY 74 PHE 108 ILE 110 TRP 115 TYR 198 ASP 228 GLY 230 THR 231THR 232 ARG 235

TABLE 5A Residues with 7 Å of the binding site for the P2₁ crystal form.GLY 11 GLN 12 GLY 13 TYR 14 LEU 30 VAL 31 ASP 32 THR 33 GLY 34 SER 35SER 36 ASN 37 VAL 69 PRO 70 TYR 71 THR 72 GLN 73 GLY 74 LYS 75 TRP 76ASP 106 LYS 107 PHE 108 PHE 109 ILE 110 TRP 115 ILE 118 GLY 120 ILE 126ALA 127 ARG 128 TYR 198 LYS 224 ILE 226 ASP 228 SER 229 GLY 230 THR 231THR 232 ASN 233 ARG 235 SER 325 THR 329 VAL 332 ALA 335

TABLE 5B Residues within 7 Å of the binding site for the C2 crystalform. LYS 9 GLY 11 GLN 12 GLY 13 TYR 14 LEU 30 ASP 32 THR 33 GLY 34 SER35 SER 36 ASN 37 VAL 69 PRO 70 TYR 71 THR 72 GLN 73 GLY 74 LYS 75 TRP 76ASP 106 LYS 107 PHE 108 PHE 109 ILE 110 TRP 115 ILE 118 ILE 126 ALA 127ARG 128 TYR 198 LYS 224 ILE 226 ASP 228 SER 229 GLY 230 THR 231 THR 232ASN 233 ARG 235 SER 325 GLN 326 THR 329 VAL 332 ALA 335

TABLE 6A Residues with 10 Å of binding site for the P2₁ crystal form.ARG 7 GLY 8 LYS 9 SER 10 GLY 11 GLN 12 GLY 13 TYR 14 TYR 15 ILE 29 LEU30 VAL 31 ASP 32 THR 33 GLY 34 SER 35 SER 36 ASN 37 PHE 38 TYR 68 VAL 69PRO 70 TYR 71 THR 72 GLN 73 GLY 74 LYS 75 TRP 76 ILE 102 SER 105 ASP 106LYS 107 PHE 108 PHE 109 ILE 110 ASN 111 SER 113 TRP 115 GLU 116 GLY 117ILE 118 LEU 119 GLY 120 LEU 121 ALA 122 TYR 123 ALA 124 GLU 125 ILE 126ALA 127 ARG 128 PRO 129 LEU 154 TRP 197 TYR 198 TYR 199 ASP 223 LYS 224SER 225 ILE 226 VAL 227 ASP 228 SER 229 GLY 230 THR 231 THR 232 ASN 233LEU 234 ARG 235 LEU 236 GLY 264 ARG 307 LYS 321 PHE 322 ALA 323 ILE 324SER 325 GLN 326 SER 327 SER 328 THR 329 GLY 330 THR 331 VAL 332 MET 333GLY 334 ALA 335 VAL 336 GLU 339

TABLE 6B Residues within 10 Å of the binding site for the C2 crystalform. ARG 7 GLY 8 LYS 9 SER 10 GLY 11 GLN 12 GLY 13 TYR 14 TYR 15 ILE 29LEU 30 VAL 31 ASP 32 THR 33 GLY 34 SER 35 SER 36 ASN 37 PHE 38 PHE 47TYR 68 VAL 69 PRO 70 TYR 71 THR 72 GLN 73 GLY 74 LYS 75 TRP 76 ILE 102SER 105 ASP 106 LYS 107 PHE 108 PHE 109 ILE 110 ASN 111 SER 113 ASN 114TRP 115 GLU 116 GLY 117 ILE 118 LEU 119 GLY 120 LEU 121 ALA 122 TYR 123ALA 124 GLU 125 ILE 126 ALA 127 ARG 128 PRO 129 LEU 154 LEU 167 VAL 170TRP 197 TYR 198 TYR 199 ASP 223 LYS 224 SER 225 ILE 226 VAL 227 ASP 228SER 229 GLY 230 THR 231 THR 232 ASN 233 LEU 234 ARG 235 LEU 236 GLY 264ARG 307 LYS 321 ALA 323 ILE 324 SER 325 GLN 326 SER 327 SER 328 THR 329GLY 330 THR 331 VAL 332 MET 333 GLY 334 ALA 335 VAL 336 GLU 339Three-Dimensional Configurations

X-ray structure coordinates define a unique configuration of points inspace. Those of skill in the art understand that a set of structurecoordinates for protein or a protein/ligand complex, or a portionthereof, define a relative set of points that, in turn, define aconfiguration in three dimensions. A similar or identical configurationcan be defined by an entirely different set of coordinates, provided thedistances and angles between coordinates remain essentially the same. Inaddition, a scalable configuration of points can be defined byincreasing or decreasing the distances between coordinates by a scalarfactor while keeping the angles essentially the same.

The present invention thus includes the scalable three-dimensionalconfiguration of points derived from the structure coordinates of atleast a portion of a human BACE molecule or molecular complex, as listedin Table 1 and Table 2, as well as structurally equivalentconfigurations, as described below. Preferably, the scalablethree-dimensional configuration includes points derived from structurecoordinates representing the locations of a plurality of the amino acidsdefining a human BACE binding pocket.

In one embodiment, the scalable three-dimensional configuration includespoints derived from structure coordinates representing the locations thebackbone atoms of a plurality of amino acids defining the human BACEbinding pocket, preferably the amino acids listed in Table 4A or 4B,more preferably the amino acids listed in Table 5A or 5B, and mostpreferably the amino acids listed in Table 6A or 6B. Alternatively, thescalable three-dimensional configuration includes points derived fromstructure coordinates representing the locations of the side chain andthe backbone atoms (other than hydrogens) of a plurality of the aminoacids defining the human BACE binding pocket, preferably the amino acidslisted in Table 4A or 4B, more preferably the amino acids listed inTable 5A or 5B, and most preferably the amino acids listed in Table 6Aor 6B.

Likewise, the invention also includes the scalable three-dimensionalconfiguration of points derived from structure coordinates of moleculesor molecular complexes that are structurally homologous to BACE, as wellas structurally equivalent configurations. Structurally homologousmolecules or molecular complexes are defined below. Advantageously,structurally homologous molecules can be identified using the structurecoordinates of human BACE according to a method of the invention.

The configurations of points in space derived from structure coordinatesaccording to the invention can be visualized as, for example, aholographic image, a stereodiagram, a model, or a computer-displayedimage, and the invention thus includes such images, diagrams or models.

Structurally Equivalent Crystal Structures

Various computational analyses can be used to determine whether amolecule or a binding pocket portion thereof is “structurallyequivalent,” defined in terms of its three-dimensional structure, to allor part of human BACE or its binding pockets. Such analyses may becarried out in current software applications, such as the MolecularSimilarity application of QUANTA (Molecular Simulations Inc., San Diego,Calif.) version 4.1, and as described in the accompanying User's Guide.

The Molecular Similarity application permits comparisons betweendifferent structures, different conformations of the same structure, anddifferent parts of the same structure. The procedure used in MolecularSimilarity to compare structures is divided into four steps: (1) loadthe structures to be compared; (2) define the atom equivalences in thesestructures; (3) perform a fitting operation; and (4) analyze theresults.

Each structure is identified by a name. One structure is identified asthe target (i.e., the fixed structure); all remaining structures areworking structures (i.e., moving structures). Since atom equivalencywithin QUANTA is defined by user input, for the purpose of thisinvention equivalent atoms are defined as protein backbone atoms (N, Cα,C, and O) for all conserved residues between the two structures beingcompared. A conserved residue is defined as a residue which isstructurally or functionally equivalent. Only rigid fitting operationsare considered.

When a rigid fitting method is used, the working structure is translatedand rotated to obtain an optimum fit with the target structure. Thefitting operation uses an algorithm that computes the optimumtranslation and rotation to be applied to the moving structure, suchthat the root mean square difference of the fit over the specified pairsof equivalent atom is an absolute minimum. This number, given inangstroms, is reported by QUANTA.

Machine Readable Storage Media

Transformation of the structure coordinates for all or a portion ofhuman BACE or the human BACE/ligand complex or one of its bindingpockets, for structurally homologous molecules as defined below, or forthe structural equivalents of any of these molecules or molecularcomplexes as defined above, into three-dimensional graphicalrepresentations of the molecule or complex can be conveniently achievedthrough the use of commercially-available software.

The invention thus further provides a machine-readable storage mediumincluding a data storage material encoded with machine readable datawhich, when using a machine programmed with instructions for using saiddata, displays a graphical three-dimensional representation of any ofthe molecule or molecular complexes of this invention that have beendescribed above. In a preferred embodiment, the machine-readable datastorage medium includes a data storage material encoded with machinereadable data which, when using a machine programmed with instructionsfor using said data, displays a graphical three-dimensionalrepresentation of a molecule or molecular complex including all or anyparts of a human BACE binding pocket or an BACE-like binding pocket, asdefined above. In another preferred embodiment, the machine-readabledata storage medium includes a data storage material encoded withmachine readable data which, when using a machine programmed withinstructions for using said data, displays a graphical three-dimensionalrepresentation of a molecule or molecular complex defined by thestructure coordinates of all of the amino acids listed in Table 1 orTable 2, ± a root mean square deviation from the backbone atoms of saidamino acids of less than about 0.65 Å.

In an alternative embodiment, the machine-readable data storage mediumincludes a data storage material encoded with a first set of machinereadable data which includes the Fourier transform of the structurecoordinates set forth in Table 1 or Table 2, and which, when using amachine programmed with instructions for using said data, can becombined with a second set of machine readable data including the x-raydiffraction pattern of a molecule or molecular complex to determine atleast a portion of the structure coordinates corresponding to the secondset of machine readable data.

For example, a system for reading a data storage medium may include acomputer including a central processing unit (“CPU”), a working memorywhich may be, e.g., RAM (random access memory) or “core” memory, massstorage memory (such as one or more disk drives or CD-ROM drives), oneor more display devices (e.g., cathode-ray tube (“CRT”) displays, lightemitting diode (“LED”) displays, liquid crystal displays (“LCDs”),electroluminescent displays, vacuum fluorescent displays, field emissiondisplays (“FEDs”), plasma displays, projection panels, etc.), one ormore user input devices (e.g., keyboards, microphones, mice, trackballs, touch pads, etc.), one or more input lines, and one or moreoutput lines, all of which are interconnected by a conventionalbidirectional system bus. The system may be a stand-alone computer, ormay be networked (e.g., through local area networks, wide area networks,intranets, extranets, or the internet) to other systems (e.g.,computers, hosts, servers, etc.). The system may also include additionalcomputer controlled devices such as consumer electronics and appliances.

Input hardware may be coupled to the computer by input lines and may beimplemented in a variety of ways. Machine-readable data of thisinvention may be inputted via the use of a modem or modems connected bya telephone line or dedicated data line. Alternatively or additionally,the input hardware may include CD-ROM drives or disk drives. Inconjunction with a display terminal, a keyboard may also be used as aninput device.

Output hardware may be coupled to the computer by output lines and maysimilarly be implemented by conventional devices. By way of example, theoutput hardware may include a display device for displaying a graphicalrepresentation of a binding pocket of this invention using a programsuch as QUANTA as described herein. Output hardware might also include aprinter, so that hard copy output may be produced, or a disk drive, tostore system output for later use.

In operation, a CPU coordinates the use of the various input and outputdevices, coordinates data accesses from mass storage devices, accessesto and from working memory, and determines the sequence of dataprocessing steps. A number of programs may be used to process themachine-readable data of this invention. Such programs are discussed inreference to the computational methods of drug discovery as describedherein. References to components of the hardware system are included asappropriate throughout the following description of the data storagemedium.

Machine-readable storage devices useful in the present inventioninclude, but are not limited to, magnetic devices, electrical devices,optical devices, and combinations thereof. Examples of such data storagedevices include, but are not limited to, hard disk devices, CD devices,digital video disk devices, floppy disk devices, removable hard diskdevices, magneto-optic disk devices, magnetic tape devices, flash memorydevices, bubble memory devices, holographic storage devices, and anyother mass storage peripheral device. It should be understood that thesestorage devices include necessary hardware (e.g., drives, controllers,power supplies, etc.) as well as any necessary media (e.g., disks, flashcards, etc.) to enable the storage of data.

Structurally Homologous Molecules, Molecular Complexes, and CrystalStructures

The structure coordinates set forth in Table 1 or Table 2 can be used toaid in obtaining structural information about another crystallizedmolecule or molecular complex. The method of the invention allowsdetermination of at least a portion of the three-dimensional structureof molecules or molecular complexes which contain one or more structuralfeatures that are similar to structural features of human BACE. Thesemolecules are referred to herein as “structurally homologous” to humanbeta secretase. Similar structural features can include, for example,regions of amino acid identity, conserved active site or binding sitemotifs, and similarly arranged secondary structural elements (e.g., αhelices and β sheets). Optionally, structural homology is determined byaligning the residues of the two amino acid sequences to optimize thenumber of identical amino acids along the lengths of their sequences;gaps in either or both sequences are permitted in making the alignmentin order to optimize the number of identical amino acids, although theamino acids in each sequence must nonetheless remain in their properorder. Preferably, two amino acid sequences are compared using theBlastp program, version 2.0.9, of the BLAST 2 search algorithm, asdescribed by Tatusova et al., FEMS Microbiol Lett 174, 247-50 (1999).Preferably, the default values for all BLAST 2 search parameters areused, including matrix=BLOSUM62; open gap penalty=11, extension gappenalty=1, gap x_dropoff=50, expect=10, wordsize=3, and filter on. Inthe comparison of two amino acid sequences using the BLAST searchalgorithm, structural similarity is referred to as “identity.”Preferably, a structurally homologous molecule is a protein that has anamino acid sequence sharing at least 65% identity with a native orrecombinant amino acid sequence of human BACE (for example, SEQ IDNO:1). More preferably, a protein that is structurally homologous tohuman BACE includes at least one contiguous stretch of at least 50 aminoacids that shares at least 80% amino acid sequence identity with theanalogous portion of the native or recombinant human BACE (for example,SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3). Methods for generatingstructural information about the structurally homologous molecule ormolecular complex are well-known and include, for example, molecularreplacement techniques.

Therefore, in another embodiment this invention provides a method ofutilizing molecular replacement to obtain structural information about amolecule or molecular complex whose structure is unknown including thesteps of:

(a) crystallizing the molecule or molecular complex of unknownstructure;

(b) generating an x-ray diffraction pattern from said crystallizedmolecule or molecular complex; and

(c) applying at least a portion of the structure coordinates set forthin Table 1 to the x-ray diffraction pattern to generate athree-dimensional electron density map of the molecule or molecularcomplex whose structure is unknown.

By using molecular replacement, all or part of the structure coordinatesof human BACE or the human BACE/ligand complex as provided by thisinvention can be used to determine the structure of a crystallizedmolecule or molecular complex whose structure is unknown more quicklyand efficiently than attempting to determine such information ab initio.

Molecular replacement provides an accurate estimation of the phases foran unknown structure. Phases are a factor in equations used to solvecrystal structures that cannot be determined directly. Obtainingaccurate values for the phases, by methods other than molecularreplacement, is a time-consuming process that involves iterative cyclesof approximations and refinements and greatly hinders the solution ofcrystal structures. However, when the crystal structure of a proteincontaining at least a structurally homologous portion has been solved,the phases from the known structure provide a satisfactory estimate ofthe phases for the unknown structure.

Thus, this method involves generating a preliminary model of a moleculeor molecular complex whose structure coordinates are unknown, byorienting and positioning the relevant portion of human BACE or thehuman BACE/modifier complex within the unit cell of the crystal of theunknown molecule or molecular complex so as best to account for theobserved x-ray diffraction pattern of the crystal of the molecule ormolecular complex whose structure is unknown. Phases can then becalculated from this model and combined with the observed x-raydiffraction pattern amplitudes to generate an electron density map ofthe structure whose coordinates are unknown. This, in turn, can besubjected to any well-known model building and structure refinementtechniques to provide a final, accurate structure of the unknowncrystallized molecule or molecular complex (Lattman, Meth. Enzymol.,115:55-77 (1985); M. G. Rossman, ed., “The Molecular ReplacementMethod,” Int. Sci. Rev. Ser., No. 13, Gordon & Breach, New York (1972)).

Structural information about a portion of any crystallized molecule ormolecular complex that is sufficiently structurally homologous to aportion of human BACE can be resolved by this method. In addition to amolecule that shares one or more structural features with human BACE asdescribed above, a molecule that has similar bioactivity, such as thesame catalytic activity, substrate specificity or ligand bindingactivity as human BACE, may also be sufficiently structurally homologousto human BACE to permit use of the structure coordinates of human BACEto solve its crystal structure.

In a preferred embodiment, the method of molecular replacement isutilized to obtain structural information about a molecule or molecularcomplex, wherein the molecule or molecular complex includes a human BACEsubunit or homolog. A “subunit” of human BACE is a human BACE moleculethat has been truncated at the N-terminus or the C-terminus, or both. Inthe context of the present invention, a “homolog” of human BACE is aprotein that contains one or more amino acid substitutions, deletions,additions, or rearrangements with respect to the amino acid sequence ofhuman BACE (SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3), but that, whenfolded into its native conformation, exhibits or is reasonably expectedto exhibit at least a portion of the tertiary (three-dimensional)structure of human BACE. For example, structurally homologous moleculescan contain deletions or additions of one or more contiguous ornoncontiguous amino acids, such as a loop or a domain. Structurallyhomologous molecules also include “modified” human BACE molecules thathave been chemically or enzymatically derivatized at one or moreconstituent amino acid, including side chain modifications, backbonemodifications, and N- and C-terminal modifications includingacetylation, hydroxylation, methylation, amidation, and the attachmentof carbohydrate or lipid moieties, cofactors, and the like.

A heavy atom derivative of human BACE is also included as a human BACEhomolog. The term “heavy atom derivative” refers to derivatives of humanBACE produced by chemically modifying a crystal of human BACE. Inpractice, a crystal is soaked in a solution containing heavy metal atomsalts, or organometallic compounds, e.g., lead chloride, goldthiomalate, thiomersal or uranyl acetate, which can diffuse through thecrystal and bind to the surface of the protein. The location(s) of thebound heavy metal atom(s) can be determined by x-ray diffractionanalysis of the soaked crystal. This information, in turn, is used togenerate the phase information used to construct three-dimensionalstructure of the protein (T. L. Blundell and N. L. Johnson, ProteinCrystallography, Academic Press (1976)).

Because human BACE can crystallize in more than one crystal form, thestructure coordinates of human BACE as provided by this invention areparticularly useful in solving the structure of other crystal forms ofhuman BACE or human BACE complexes.

The structure coordinates of human BACE as provided by this inventionare particularly useful in solving the structure of human BACE mutants.Mutants may be prepared, for example, by expression of human BACE cDNApreviously altered in its coding sequence by oligonucleotide-directedmutagenesis. Mutants may also be generated by site-specificincorporation of unnatural amino acids into BACE proteins using thegeneral biosynthetic method of Noren et al., Science, 244:182-88 (1989).In this method, the codon encoding the amino acid of interest inwild-type human BACE is replaced by a “blank” nonsense codon, TAG, usingoligonucleotide-directed mutagenesis. A suppressor tRNA directed againstthis codon is then chemically aminoacylated in vitro with the desiredunnatural amino acid. The aminoacylated tRNA is then added to an invitro translation system to yield a mutant human BACE with thesite-specific incorporated unnatural amino acid.

Selenocysteine or selenomethionine may be incorporated into wild-type ormutant human BACE by expression of human BACE-encoding cDNAs inauxotrophic E. coli strains (Hendrickson et al., EMBO J., 9:1665-72(1990)). In this method, the wild-type or mutagenized human BACE cDNAmay be expressed in a host organism on a growth medium depleted ofeither natural cysteine or methionine (or both) but enriched inselenocysteine or selenomethionine (or both). Alternatively,selenomethionine analogues may be prepared by down regulation methioninebiosynthesis. (Benson et al., Nat. Struct. Biol., 2:644-53 (1995); VanDuyne et al., J. Mol. Biol., 229:105-24 (1993)).

The structure coordinates of human BACE listed in Table 1 and Table 2are also particularly useful to solve the structure of crystals of humanBACE, human BACE mutants or human BACE homologs co-complexed with avariety of chemical entities. This approach enables the determination ofthe optimal sites for interaction between chemical entities, includingcandidate human BACE modifiers and human BACE. Potential sites formodification within the various binding sites of the molecule can alsobe identified. This information provides an additional tool fordetermining the most efficient binding interactions, for example,increased hydrophobic interactions, between human BACE and a chemicalentity. For example, high resolution x-ray diffraction data collectedfrom crystals exposed to different types of solvent allows thedetermination of where each type of solvent molecule resides. Smallmolecules that bind tightly to those sites can then be designed andsynthesized and tested for their potential human BACE inhibitionactivity.

All of the complexes referred to above may be studied using well-knownx-ray diffraction techniques and may be refined versus 1.5-3.5 Åresolution x-ray data to an R value of about 0.30 or less using computersoftware, such as X-PLOR (Yale University, 81992, distributed byMolecular Simulations, Inc.; see, e.g., Blundell & Johnson, supra; Meth.Enzymol., Vol. 114 & 115, H. W. Wyckoff et al., eds., Academic Press(1985)). This information may thus be used to optimize known human BACEmodifiers, and more importantly, to design new human BACE modifiers.

The invention also includes the unique three-dimensional configurationdefined by a set of points defined by the structure coordinates for amolecule or molecular complex structurally homologous to human BACE asdetermined using the method of the present invention, structurallyequivalent configurations, and magnetic storage media including such setof structure coordinates.

Further, the invention includes structurally homologous molecules asidentified using the method of the invention.

Homology Modeling

Using homology modeling, a computer model of a human BACE homolog can bebuilt or refined without crystallizing the homolog. First, a preliminarymodel of the human BACE homolog is created by sequence alignment withhuman BACE, secondary structure prediction, the screening of structurallibraries, or any combination of those techniques. Computationalsoftware may be used to carry out the sequence alignments and thesecondary structure predictions. Structural incoherences, e.g.,structural fragments around insertions and deletions, can be modeled byscreening a structural library for peptides of the desired length andwith a suitable conformation. For prediction of the side chainconformation, a side chain rotamer library may be employed. If the humanBACE homolog has been crystallized, the final homology model can be usedto solve the crystal structure of the homolog by molecular replacement,as described above. Next, the preliminary model is subjected to energyminimization to yield an energy minimized model. The energy minimizedmodel may contain regions where stereochemistry restraints are violated,in which case such regions are remodeled to obtain a final homologymodel. The homology model is positioned according to the results ofmolecular replacement, and subjected to further refinement includingmolecular dynamics calculations.

Rational Drug Design

Computational techniques can be used to screen, identify, select and/ordesign chemical entities capable of associating with human BACE orstructurally homologous molecules. Knowledge of the structurecoordinates for human BACE permits the design and/or identification ofsynthetic compounds and/or other molecules which have a shapecomplementary to the conformation of the human BACE binding site. Inparticular, computational techniques can be used to identify or designchemical entities, such as inhibitors, agonists and antagonists, thatassociate with a human BACE binding pocket or an BACE-like bindingpocket. Potential modifiers may bind to or interfere with all or aportion of an active site of human BACE, and can be competitive,non-competitive, or uncompetitive inhibitors; or interfere withdimerization by binding at the interface between the two monomers. Onceidentified and screened for biological activity, theseinhibitors/agonists/antagonists may be used therapeutically orprophylactically to block human BACE activity and, thus, prevent theonset and/or further progression of Alzheimer's disease.Structure-activity data for analogues of ligands that bind to orinterfere with human BACE or BACE-like binding pockets can also beobtained computationally.

The term “chemical entity,” as used herein, refers to chemicalcompounds, complexes of two or more chemical compounds, and fragments ofsuch compounds or complexes. Chemical entities that are determined toassociate with human BACE are potential drug candidates. Data stored ina machine-readable storage medium that displays a graphicalthree-dimensional representation of the structure of human BACE or astructurally homologous molecule, as identified herein, or portionsthereof may thus be advantageously used for drug discovery. Thestructure coordinates of the chemical entity are used to generate athree-dimensional image that can be computationally fit to thethree-dimensional image of human BACE or a structurally homologousmolecule. The three-dimensional molecular structure encoded by the datain the data storage medium can then be computationally evaluated for itsability to associate with chemical entities. When the molecularstructures encoded by the data is displayed in a graphicalthree-dimensional representation on a computer screen, the proteinstructure can also be visually inspected for potential association withchemical entities.

One embodiment of the method of drug design involves evaluating thepotential association of a known chemical entity with human BACE or astructurally homologous molecule, particularly with a human BACEbinding, pocket or BACE-like binding pocket. The method of drug designthus includes computationally evaluating the potential of a selectedchemical entity to associate with any of the molecules or molecularcomplexes set forth above. This method includes the steps of: (a)employing computational means to perform a fitting operation between theselected chemical entity and a binding pocket or a pocket nearby thebinding pocket of the molecule or molecular complex; and (b) analyzingthe results of said fitting operation to quantify the associationbetween the chemical entity and the binding pocket.

In another embodiment, the method of drug design involvescomputer-assisted design of chemical entities that associate with humanBACE, its homologs, or portions thereof. Chemical entities can bedesigned in a step-wise fashion, one fragment at a time, or may bedesigned as a whole or “de novo.”

To be a viable drug candidate, the chemical entity identified ordesigned according to the method must be capable of structurallyassociating with at least part of a human BACE or BACE-like bindingpockets, and must be able, sterically and energetically, to assume aconformation that allows it to associate with the human BACE orBACE-like binding pocket. Non-covalent molecular interactions importantin this association include hydrogen bonding, van der Waalsinteractions, hydrophobic interactions, and electrostatic interactions.Conformational considerations include the overall three-dimensionalstructure and orientation of the chemical entity in relation to thebinding pocket, and the spacing between various functional groups of anentity that directly interact with the BACE-like binding pocket orhomologs thereof.

Optionally, the potential binding of a chemical entity to a human BACEor BACE-like binding pocket is analyzed using computer modelingtechniques prior to the actual synthesis and testing of the chemicalentity. If these computational experiments suggest insufficientinteraction and association between it and the human BACE or BACE-likebinding pocket, testing of the entity is obviated. However, if computermodeling indicates a strong interaction, the molecule may then besynthesized and tested for its ability to bind to or interfere with ahuman BACE or BACE-like binding pocket. Binding assays to determine if acompound (e.g., an inhibitor) actually interferes with human BACE canalso be performed and are well known in the art. Binding assays mayemploy kinetic or thermodynamic methodology using a wide variety oftechniques including, but not limited to, microcalorimetry, circulardichroism, capillary zone electrophoresis, nuclear magnetic resonancespectroscopy, fluorescence spectroscopy, and combinations thereof.

One method for determining whether a modifier binds to a protein isisothermal denaturation. This method includes taking a sample of aprotein (in the presence or absence of substrates) at a fixed elevatedtemperature where denaturation of the protein occurs in a given timeframe, adding the chemical entity to the protein, and monitoring therate of denaturation. If the chemical entity does bind to the protein,it is expected that the rate of denaturation would be slower in thepresence of the chemical entity than in the absence of the chemicalentity. For example, this method has been described in Epps et al.,Anal. Biochem., 292:40-50 (2001).

One skilled in the art may use one of several methods to screen chemicalentities or fragments for their ability to associate with a human BACEor BACE-like binding pocket. This process may begin by visual inspectionof, for example, a human BACE or BACE-like binding pocket on thecomputer screen based on the human BACE structure coordinates listed inTable 1 or other coordinates which define a similar shape generated fromthe machine-readable storage medium. Selected fragments or chemicalentities may then be positioned in a variety of orientations, or docked,within the binding pocket. Docking may be accomplished using softwaresuch as QUANTA and SYBYL, followed by energy minimization and moleculardynamics with standard molecular mechanics forcefields, such as CHARMMand AMBER.

Specialized computer programs may also assist in the process ofselecting fragments or chemical entities. Examples include GRID(Goodford, J. Med. Chem., 28:849-57 (1985); available from OxfordUniversity, Oxford, UK); MCSS (Miranker et al., Proteins: Struct. Funct.Gen., 11:29-34 (1991); available from Molecular Simulations, San Diego,Calif.); AUTODOCK (Goodsell et al., Proteins: Struct. Funct. Genet.,8:195-202 (1990); available from Scripps Research Institute, La Jolla,Calif.); and DOCK (Kuntz et al., J. Mol. Biol., 161:269-88 (1982);available from University of California, San Francisco, Calif.).

Once suitable chemical entities or fragments have been selected, theycan be assembled into a single compound or complex. Assembly may bepreceded by visual inspection of the relationship of the fragments toeach other on the three-dimensional image displayed on a computer screenin relation to the structure coordinates of human BACE. This would befollowed by manual model building using software such as QUANTA or SYBYL(Tripos Associates, St. Louis, Mo.).

Useful programs to aid one of skill in the art in connecting theindividual chemical entities or fragments include, without limitation,CAVEAT (P. A. Bartlett et al., in “Molecular Recognition: Chemical andBiological Problems,” Special Publ., Royal Chem. Soc., 78:182-96 (1989);Lauri et al., J. Comput. Aided Mol. Des. 8:51-66 (1994); available fromthe University of California, Berkeley, Calif.); 3D database systemssuch as ISIS (available from MDL Information Systems, San Leandro,Calif.; reviewed in Martin, J. Med. Chem., 35:2145-54 (1992)); and HOOK(Eisen et al., Proteins: Struc., Funct., Genet., 19:199-221 (1994);available from Molecular Simulations, San Diego, Calif.).

Human BACE binding compounds may be designed “de novo” using either anempty binding site or optionally including some portion(s) of a knownmodifier(s). There are many de novo ligand design methods including,without limitation, LUDI (Böhm, J. Comp. Aid. Molec. Design, 6:61-78(1992); available from Molecular Simulations Inc., San Diego, Calif.);LEGEND (Nishibata et al., Tetrahedron, 47:8985-90 (1991); available fromMolecular Simulations Inc., San Diego, Calif.); LeapFrog (available fromTripos Associates, St. Louis, Mo.); and SPROUT (Gillet et al., J.Comput. Aided Mol. Design, 7:127-53 (1993); available from theUniversity of Leeds, UK).

Once a compound has been designed or selected by the above methods, theefficiency with which that entity may bind to or interfere with a humanBACE or BACE-like binding pocket may be tested and optimized bycomputational evaluation. For example, an effective human BACE orBACE-like binding pocket modifier must preferably demonstrate arelatively small difference in energy between its bound and free states(i.e., a small deformation energy of binding). Thus, the most efficienthuman BACE or BACE-like binding pocket modifiers should preferably bedesigned with a deformation energy of binding of at most about 10kcal/mole; more preferably, at most 7 kcal/mole. Human BACE or BACE-likebinding pocket modifiers may interact with the binding pocket in morethan one conformation that is similar in overall binding energy. Inthose cases, the deformation energy of binding is taken to be thedifference between the energy of the free entity and the average energyof the conformations observed when the modifier binds to the protein.

An entity designed or selected as binding to or interfering with a humanBACE or BACE-like binding pocket may be further computationallyoptimized so that in its bound state it would preferably lack repulsiveelectrostatic interaction with the target enzyme and with thesurrounding water molecules. Such non-complementary electrostaticinteractions include repulsive charge-charge, dipole-dipole, andcharge-dipole interactions.

Specific computer software is available in the art to evaluate compounddeformation energy and electrostatic interactions. Examples of programsdesigned for such uses include: Gaussian 94, revision C (M. J. Frisch,Gaussian, Inc., Pittsburgh, Pa. 81995); AMBER, version 4.1 (P. A.Kollman, University of California at San Francisco, 81995);QUANTA/CHARMM (Molecular Simulations, Inc., San Diego, Calif. 81995);Insight II/Discover (Molecular Simulations, Inc., San Diego, Calif.81995); DelPhi (Molecular Simulations, Inc., San Diego, Calif. 81995);and AMSOL (Quantum Chemistry Program Exchange, Indiana University).These programs may be implemented, for instance, using a SiliconGraphics workstation such as an Indigo² with “IMPACT” graphics. Otherhardware systems and software packages will be known to those skilled inthe art.

Another approach encompassed by this invention is the computationalscreening of small molecule databases for chemical entities or compoundsthat can bind in whole, or in part, to a human BACE or BACE-like bindingpocket. In this screening, the quality of fit of such entities to thebinding site may be judged either by shape complementarity or byestimated interaction energy (Meng et al., J. Comp. Chem., 13:505-24(1992)).

This invention also enables the development of chemical entities thatcan isomerize to short-lived reaction intermediates in the chemicalreaction of a substrate or other compound that interferes with or withhuman BACE. Time-dependent analysis of structural changes in human BACEduring its interaction with other molecules is carried out. The reactionintermediates of human BACE can also be deduced from the reactionproduct in co-complex with human BACE. Such information is useful todesign improved analogues of known human BACE modifiers or to designnovel classes of modifiers based on the reaction intermediates of thehuman BACE and modifier co-complex. This provides a novel route fordesigning human BACE modifiers with both high specificity and stability.

Yet another approach to rational drug design involves probing the humanBACE crystal of the invention with molecules including a variety ofdifferent functional groups to determine optimal sites for interactionbetween candidate human BACE modifiers and the protein. For example,high resolution x-ray diffraction data collected from crystals soaked inor co-crystallized with other molecules allows the determination ofwhere each type of solvent molecule sticks. Molecules that bind tightlyto those sites can then be further modified and synthesized and testedfor their BACE modifier activity (Travis, Science, 262:1374 (1993)).

In a related approach, iterative drug design is used to identifymodifiers of human BACE. Iterative drug design is a method foroptimizing associations between a protein and a compound by determiningand evaluating the three-dimensional structures of successive sets ofprotein/compound complexes. In iterative drug design, crystals of aseries of protein/compound complexes are obtained and then thethree-dimensional structures of each complex is solved. Such an approachprovides insight into the association between the proteins and compoundsof each complex. This is accomplished by selecting compounds withinhibitory activity, obtaining crystals of this new protein/compoundcomplex, solving the three-dimensional structure of the complex, andcomparing the associations between the new protein/compound complex andpreviously solved protein/compound complexes. By observing how changesin the compound affected the protein/compound associations, theseassociations may be optimized.

A compound that is identified or designed as a result of any of thesemethods can be obtained (or synthesized) and tested for its biologicalactivity, e.g., inhibition of BACE activity.

Assay systems that can be used to demonstrate efficacy of the compoundinhibitors of the invention are known. Representative assay systems aredescribed, for example, in U.S. Pat. Nos. 5,942,400 (Anderson et al.)and 5,744,346 (Chrysler et al.).

Pharmaceutical Compositions (Modifiers)

Pharmaceutical compositions of this invention include a potentialmodifier of human BACE activity identified according to the invention,or a pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier, adjuvant, or vehicle. The term “pharmaceuticallyacceptable carrier” refers to a carrier(s) that is “acceptable” in thesense of being compatible with the other ingredients of a compositionand not deleterious to the recipient thereof. Optionally, the pH of theformulation is adjusted with pharmaceutically acceptable acids, bases,or buffers to enhance the stability of the formulated compound or itsdelivery form.

Methods of making and using such pharmaceutical compositions are alsoincluded in the invention. The pharmaceutical compositions of theinvention can be administered orally, parenterally, by inhalation spray,topically, rectally, nasally, buccally, vaginally, or via an implantedreservoir. Oral administration or administration by injection ispreferred. The term parenteral as used herein includes subcutaneous,intracutaneous, intravenous, intramuscular, intra-articular,intrasynovial, intrasternal, intrathecal, intralesional, andintracranial injection or infusion techniques.

Dosage levels of about 0.01 to about 100 mg/kg body weight per day,preferably about 0.5 to about 75 mg/kg body weight per day of the humanBACE inhibitory compounds described herein are useful for the preventionand treatment of human BACE mediated disease. Typically, thepharmaceutical compositions of this invention will be administered about1 to about 5 times per day or alternatively, as a continuous infusion.Such administration can be used as a chronic or acute therapy. Theamount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. A typicalpreparation will contain about 5% to about 95% active compound (w/w).Preferably, such preparations contain about 20% to about 80% activecompound.

In order that this invention be more fully understood, the followingexamples are set forth. These examples are for the purpose ofillustration only and are not to be construed as limiting the scope ofthe invention in any way.

EXAMPLES Example 1 Crystallization and Structure Determination of HumanBACE in P2₁, Crystal Form

Expression, Purification, and Crystallization

Two BACE constructs, 1) pET11a-T7.Tag-Gly-Ser-Met-(A⁻GV . . . QTDES)(referred to as pET11a-BACE; SEQ ID NO:1) and 2)pQE80L-Met-Arg-Gly-Ser-(His)₆-Gly-Ser-Ile-Glu-Thr-Asp-(TQH . . . QTDES)(referred to as pQE80L-BACE; SEQ ID NO:2) were cloned and expressed asinclusion bodies. Inclusion bodies obtained from 10 liters of cellculture were washed one time in 10 mM TRIS buffer (pH 8.12) and 1 mMEDTA (TE). The inclusion bodies were extracted with 15-20 ml 8 M urea,100 mM AMPSO, 1 mM glycine, 1 mM EDTA, and 100 mM β-Mercaptoethanol(BME, pH 10.5-10.8). After centrifugation, the protein concentration ofthe supernatant was adjusted by dilution with the above buffer to readapproximately 5.0 at A₂₈₀. The protein was then diluted with 8 M urea,100 mM AMPSO, 1 mM glycine, 1 mM EDTA, and the BME concentrationadjusted to 10 mM by the addition of solid BME to read an A₂₈₀˜0.5 andpH 10.5-10.8. The solution was centrifuged. The refolding worked bestwith AMPSO, however, the AMPSO could be substituted with CAPS or TRIS.Analysis of the sample in 8 M urea by SDS-PAGE revealed BACE as themajor component of the solubilized inclusion bodies. BACE migrated as aband of Mr=50,000. Refolding was carried out by a 20-25 fold dilutionwith cold water (4-15° C.). Upon dilution, the pH dropped automaticallyto 9.5-10.2. The sample was then allowed to rest in the cold room.Activity assays were performed daily to monitor protein refolding.Results from various experiments indicated that maximal activity wasusually reached at day 5.

The pH of the refolded protein was lowered from about 10 to 8.5 with HCland loaded onto a Q-Sepharose column (5.0 cm×2.8 cm). BACE was elutedwith 0.75 M NaCl in 0.4 M Urea and 10 mM TRIS buffer (pH 8.2). Afterconcentration, BACE was delivered onto a Sepbacryl-S200 column (2.5cm×130 cm) equilibrated and eluted in 20 mM Hepes buffer and 100 mM NaCl(pH 8.0). This molecular sieving step resolved the active monomeric BACEfrom its aggregated forms. Finally, the sample was brought to pH 4.5 andapplied to a 10 ml affinity column equilibrated at the same pH. Thecolumn was washed with 6 column volumes of 20 mM sodium acetate buffer(pH 4.5) and 150 mM NaCl. BACE was eluted at pH 8.5 in 0.1 M boratebuffer. The resin had been cross-linked with the synthetic peptide shownin FIG. 2. This final step removed any residual contaminants. From 10liter of E. Coli cell culture, the amount of protein obtained was 55 mg,and 93 mg of highly purified pET11a-BACE and pQE-80-BACE constructs,respectively.

Purified pET11a-BACE or pQE80L-BACE was dialyzed into 100 mM sodiumborate (pH 8.5). Initial attempts to crystallize this material byincubation of inhibitors in a sparse matrix screen were unsuccessful.Dynamic light scattering data indicated that there was an aggregatespecies present that might interfere with crystallization. In an effortto reduce non-specific aggregation, the enzyme was concentrated in thepresence of the inhibitor shown in FIG. 1. The purpose of this wastwo-fold: first, the compound would bind active molecules forming ahomogenous population, and second, the complex, once locked in placewould then not be able to contribute to non-specific aggregation. Theconcentration of the stock protein solution was determined andmultiplied by 2.4. This calculation provided the excess inhibitor to beadded to the dilute protein sample before the concentration. Theappropriate amount of 50 mM inhibitor stock solution (in 100% DMSO) wasadded, and the solution was incubated on ice for about 30 minutes toabout 60 minutes before concentration. A 30K MWCO Ultrafree-4concentrator (Millipore, Bedford, Mass.) was used with a pretreatmentusing 2.0 ml of the following solution: 20 mM Hepes (pH 7.8), 20%Glycerol, 5% PEG 8000, 0.1 M NaCl. The sample was spun at 3810 rpm(3000×g) in an SH-3000 rotor in 10 minute increments until desiredvolume was achieved. The membrane was rinsed with 2×1.0 ml 20 mM Hepes(pH 7.8). The first aliquot of protein:compound mix was added to theconcentrator and spun as above until ½ the volume remained. Theconcentrator was gently inverted to mix protein, and another aliquot wasadded. The above procedure was repeated until all of the unconcentratedprotein:compound mix was in the concentrator. At this point, the samplewas gently concentrated until final volume was reached that would giveapproximately 20 mg/ml concentration. The concentrated sample was usedfor co-crystallization studies.

Sparse matrix screening using the commercially available Hampton Screen1 (Hampton Research, Laguna Nigel, Calif.) and Wizard 1 screens (EmeraldBiostructures, Bainbridge Island, Wash.) was performed using the hangingdrop method. Crystals were obtained in Hampton Screen 1, condition 11(1.0 M ammonium phosphate and 0.1 M sodium citrate (pH 5.6)). Crystalswere lozenge shaped, twinned, and had dimensions of approximately 0.15mm-0.2 mm×0.1 mm-0.15 mm. The crystals stained positive for protein withIzit dye (Hampton Research, Laguna Nigel, Calif.). No other crystalswere obtained from this screen. Initial optimization experimentsresulted in crystals that possessed sharp and well-defined edges.Crystals here were still slightly twinned, and of approximately 0.2mm-0.25 mm×0.1 mm-0.2 mm in size. Further optimization with a focusedscreen yielded single crystals in trapezoidal morphology, withapproximate dimensions of 0.25 mm-0.4 mm×0.15 mm-0.25 mm. The next setof optimization experiments introduced ammonium citrate as a buffersystem in the range of pH 4.5-6.1. These solutions, along with carefuloptimization of ammonium phosphate concentration yielded single crystalsin the 0.35 mm×0.2 mm size, which diffracted to 2.5 Å on an X-raysource. The crystals grew in 0.7 M ammonium phosphate and 0.1 M ammoniumcitrate (pH 4.71). For cryogenic experiments, synthetic mother liquorbased on the well solution where the drops crystallized was preparedwith a range of DMSO or glycerol. A typical cryo solution was: 0.7 Mammonium phosphate, 0.05 M ammonium citrate (pH 4.71), 0.05 M sodiumborate (pH 8.5), and 5-30% glycerol in 5% increments. Attempts to loopout single crystals for soaking experiments resulted in the eventualcracking of the crystal. Experiments where the cryo solution was addedto the crystallization drop in a stepwise, incremental fashion over a 3minute incubation time resulted in crystals soaked into 25% glycerol.The crystal was then flash frozen in liquid nitrogen and held in astorage Dewar until time for data collection on the X-ray source.

X-Ray Diffraction Characterization

Initial data collection was carried out at the Advanced Photon Source(Argonne, Ill.) at beamline 17-ID. These initial crystals were verysmall, but diffracted to 3.5 Å using synchrotron radiation. Subsequentdata collection was carried out on another X-ray source (a Rigaku RUH3RX-ray generator using osmic confocal mirros) with a R-axis IV++ detector(Molecular Structure Corporation, The Woodlands, Tx). Optimized crystals(significantly larger than the initial ones tested at the synchrotron)diffracted to 2.5 Å. Crystals were of the space group P2₁, with celldimensions of a=81±20 Å, b=103±20 Å, c=100±20 Å, α=γ=90°, and β=105±10°.The Matthews coefficient for these crystals, assuming that there arethree molecules in the asymmetric unit, is 2.7 Å/Da with 54% solvent.The structure determination (see below) revealed the presence ofelectron density in the active site appropriate for the inhibitor shownin FIG. 1.

Molecular Replacement

A molecular replacement solution was determined using AMORE (Navaza,Acta Cryst., D50:157-63 (1994); Collaborative Computational Project N4,Acta Cryst. D50:760-63 (1994))) by utilizing a previously solvedstructure of human BACE from CHO crystals. The initial rotation solutiongave three strong peaks of 11.7σ, 11.1σ, and 10.8σ The presence of thethree strong peaks suggested that three molecules might be present inthe asymmetric unit. A translation search in space group P2₁, resultedin a correlation coefficient of 33.2 with an R-factor of 47.8% to 4 Åresolution for the first molecule. A translation search for the secondmolecule (keeping the first molecule fixed) resulted in an improvedcorrelation coefficient of 42.8 with an R-factor of 43.6% to 4 Åresolution for both molecules. A translation search for the thirdmolecule (keeping the first two molecules fixed) resulted in an improvedcorrelation coefficient of 63.8 with an R-factor of 35.3% to 4 Åresolution for all three molecules.

TABLE 7 Data collection statistics for initial data set of Human BACEformed from E. coli (pET11a-BACE) produced protein used for the intialmolecular replacement solution (data collected at λ 1.0000 Å at APS,17-ID). Data processed with HKL2000. Shell limit Lower Upper AverageAverage Norm. Linear Square Angstrom I error stat. Chi**2 R-fac R-fac50.00  6.12 1967.3 130.2 117.8 0.731 0.051 0.050 6.12 4.86 1310.8 187.1183.3 0.344 0.079 0.084 4.86 4.24 1629.5 237.3 232.5 0.384 0.085 0.0814.24 3.85 1015.3 264.6 262.7 0.324 0.139 0.123 3.85 3.58 744.0 289.1288.1 0.296 0.198 0.183 3.58 3.37 407.7 297.4 297.1 0.253 0.328 0.2903.37 3.20 256.3 300.3 300.1 0.223 0.460 0.385 3.20 3.06 160.3 311.7311.6 0.222 0.649 0.530 3.06 2.94 152.7 343.0 342.9 0.232 0.689 0.5372.94 2.84 293.8 318.0 317.3 0.424 0.556 0.515 All reflections 934.4253.8 250.8 0.362 0.126 0.100

TABLE 8 Data collection statistics for 2.15 Å resolution data set ofHuman BACE formed from E. coli (pET11a-BACE) produced protein used forrefinement (data collected at λ 1.54 Å on home source X-rays). Dataprocessed with D*trek. Rmerge vs Resolution Resolution Average Num NumNum Num <<I>/ ChiSq Rmerge Rmerge range counts obs rejs ovlps mults<sig>> norm shell cumul 19.92-4.61  11176 42087 465 41527 8927 16.2 0.670.034 0.034 4.61-3.67 11419 41602 267 41285 8845 15.2 0.68 0.037 0.0353.67-3.21 5995 41314 334 40922 8797 11.5 0.91 0.052 0.039 3.21-2.92 295241070 399 40606 8752 8.4 1.10 0.074 0.042 2.92-2.71 1601 40643 545 400058654 6.0 1.28 0.106 0.045 2.71-2.55 981 40599 683 39773 8615 4.4 1.310.142 0.048 2.55-2.42 630 40344 731 39480 8578 3.5 1.24 0.181 0.0502.42-2.32 430 40120 685 39268 8515 2.9 1.08 0.220 0.052 2.32-2.23 33539822 647 39030 8481 2.5 0.97 0.255 0.054 2.23-2.15 221 39729 374 392498521 2.2 0.76 0.310 0.055 19.92-2.15  3655 407330 5130 401145 86685 7.41.00 0.055 0.055 Redundancy vs Resolution Resolution Calc Percent ofreflections measured N times, N = % Comp % Comp range unique 0 1 2 3 45-8 9-12 >12 shell cumul 19.92-4.61  9021 0.0 1.1 6.4 2.4 32.3 57.8 0.00.0 100.0 100.0 4.61-3.67 8896 0.0 0.6 4.7 3.4 27.2 64.1 0.0 0.0 100.0100.0 3.67-3.21 8880 0.3 0.7 4.7 4.0 25.1 65.3 0.0 0.0 99.7 99.93.21-2.92 8856 0.4 0.7 4.9 4.3 24.1 65.5 0.0 0.0 99.6 99.8 2.92-2.718817 0.8 1.1 5.2 4.6 23.4 64.9 0.0 0.0 99.2 99.7 2.71-2.55 8862 1.2 1.65.2 4.8 22.9 64.4 0.0 0.0 98.8 99.6 2.55-2.42 8820 1.2 1.5 5.6 5.0 22.664.1 0.0 0.0 98.8 99.4 2.42-2.32 8822 1.6 1.9 5.3 5.0 22.7 63.6 0.0 0.098.4 99.3 2.32-2.23 8800 2.0 1.6 5.6 5.0 22.8 63.0 0.0 0.0 98.0 99.22.23-2.15 8831 2.3 1.2 5.6 4.8 23.2 62.9 0.0 0.0 97.7 99.0 19.92-2.15 88605 1.0 1.2 5.3 4.3 24.6 63.5 0.0 0.0 99.0 99.0Model Building and Refinement

Further rigid body refinement of the model in CNX (MolecularSimulations, Inc) followed by minimization refinement gave an R-factorof 28.9% and a Free R-factor of 33.2% to 3.4 | with an overall B-factorof 33.3 Å². The subsequent availability of a higher resolution data setto 2.15 Å afforded the opportunity to conduct a suitable refinement ofthe structure. The refinement against higher resolution data wasinitiated with a rigid body refinement followed by minimization andB-factor refinement leading to a R-factor of 31.6% and a Free R-factorof 34.8%. During each cycle of refinement a bulk solvent correction wasincorporated (Jiang et al., J. Mol. Biol., 243:100-15 (1994)). Progressof the refinement was monitored by a decrease in both the R-factor andFree R-factor.

At this point, inspection of the electron density map within the activesite revealed electron density that was unaccounted for by the proteinmodel and consistent with the shape of the inhibitor shown in FIG. 1that was present in the crystallization conditions. Model building wasdone using the program CHAIN (Sack, Journal of Molecular Graphics,6:224-25 (1988)) and LORE (Finzel, Meth. Enzymol., 277:230-42 (1997)).Rebuilding of the model and the addition of water molecules into themodel using the 2.15 Å resolution map afforded the opportunity forfurther cycles of refinement (including the inhibitor) givingimprovement of the R-factor to 24.5% and a Free R-factor of 27.3%. Themodel includes three residues from the N terminal pro-region (61P-63P),residues 1-157, 169-309, and 316-385. Loops for residues 158-168 and310-316 were disordered in the electron density and therefore have beenomitted from the model.

TABLE 9 Refinement Statistics for structure of Human BACE from (pET11a-BACE) No. of R-factor Free R-factor reflections 20-2.15 Å 0.245 0.27387560 F ≧ 2σ Bonds (Å) Angles (°) r.m.s deviation from 0.009 1.5 idealgeometry Number of atoms Average B-factor Protein 8790 36.6 Waters 25835.3 Ligand 123 31.4 Total 9171 36.5

Example 2 Crystallization and Structure Determination of Human BACE inC2 Crystal Form

Expression, Purification, and Crystallization

A third BACE construct, pQE70-Met-Arg-Gly-Ser-Phe-Val-Glu- . . .Thr-Asp-Glu-Ser-Arg-Ser-(His)₆ (see SEQ ID NO:3) referred to asPQE70-BACE was cloned and expressed as inclusion bodies. Inclusionbodies obtained from 40 liters of cell culture were washed one time in700 ml of 10 mM TRIS buffer, pH 8.12, 1 mM EDTA (TE). The inclusionbodies were extracted with 400 ml 7.5 M urea, 100 mM AMPSO, 1 mMglycine, 1 mM EDTA, and 100 mM β-Mercaptoethanol (BME), pH 10.5-10.8.After centrifugation, the protein concentration of the supernatant wasadjusted by dilution with the above buffer to read ˜5.0 at A₂₈₀. Theprotein was then diluted with 7.5 M urea, 100 mM AMPST, 1 mM glycine, 1mM EDTA, and the BME concentration adjusted to 10 mM by the addition ofBME to read an A₂₈₀˜0.5 and a pH=10.5-10.8. The solution wascentrifuged. Analysis of the sample in 7.5 M urea by SDS-PAGE revealedBACE as the major component of the solubilized inclusion bodies. BACEmigrated as a band of Mr˜45,000. Refolding was carried out by a 20-25fold dilution with cold water (4-15° C.). Upon dilution, the pH droppedautomatically to 9.5-10.2. The sample was then allowed to rest in thecold room. Activity assays were performed daily to monitor proteinrefolding. Results from various experiments indicated that maximalactivity was usually reached after 4-5 weeks.

Prior to purification, the pH of the refolded protein was lowered fromabout 10 to 8.5 with HCl. The solution was loaded onto three 50 mlQ-Sepharose columns (Pharmacia Biotech XK 50). The columns werepre-equilibrated in 0.4 M Urea, 10 mM AMPSO, pH 8.5. After refoldedprotein was loaded onto the columns, they were washed with 500 ml of 0.4M Urea, 10 mM TRIS, pH 8.2. The columns were eluted with 180-245 ml of0.75 M NaCl in 0.4 M Urea 10 mM TRIS buffer, pH 8.2. The eluates werethen dialyzed versus 20 mM HEPES, pH 8.0. The samples were then removedfrom dialysis and dropped into 1 M NaMES, pH 5.7 (0.1 M finalconcentration). After centrifugation (20K×g) the supernatant was droppedinto 1 M Na-acetate, 1 M NaMES, pH 5.0 (0.2 M Na-acetate, 0.28 M Na-MESwas the final concentration). No precipitation was observed at thisstep. This solution was then applied to a 15 ml affinity columnequilibrated at the same pH. The column was washed with 6 column volumesof 20 mM sodium acetate buffer pH 4.5, 150 mM NaCl. BACE was eluted atpH 8.5 using about 50 ml of 0.1 M borate buffer. The resin had beencross-linked with the synthetic peptide shown in FIG. 2. This final stepremoved any residual contaminants. From 40 liter of E. coli cellculture, the amount of protein obtained was 137 mg of highly purifiedpQE70-BACE construct. Purified pQE70-BACE was dialyzed into 100 mMNaBorate pH 8.5.

In an effort to reduce non-specific aggregation, the enzyme wasconcentrated in the presence of the inhibitor. The purpose of this wastwo-fold: First, the compound would bind active molecules forming ahomogenous population, and second, the complex, once locked in placewould then not be able to contribute to non-specific aggregation. Theconcentration of the stock protein solution was determined andmultiplied by 2.4. This calculation provides the excess inhibitor to beadded to the dilute protein sample before the concentration. Theappropriate amount of 50 mM inhibitor stock solution (in 100% DMSO) wasadded, and the solution was incubated on ice 30 minutes beforeconcentration. A 30K MWCO (Molecular Weight Cut-Off) Ultrafree-4concentrator (Millipore, Bedford, Mass.) was pretreated with 2.0 ml ofthe following solution: 20 mM Hepes pH 7.8, 20% Glycerol, 5% PEG 8000,0.1 M NaCl. The sample was spun at 3810 rpm (3000×g) in an SH-3000 rotorin 10 minute increments until desired volume is achieved. The membranewas rinsed with 2×1.0 ml 20 mM Hepes pH 7.8. The first aliquot ofprotein:compound mix was added to the concentrator and spun as aboveuntil ½ the volume remained. The concentrator was gently inverted to mixthe protein and another aliquot was added. The above procedure wasrepeated until all of the unconcentrated protein:compound mix was in theconcentrator. At this point, the sample was gently concentrated until afinal volume was reached that yielded approx. 8-10 mg/ml concentration.This concentrated sample was used for co-crystallization studies. It wasalso determined that concentrating the protein in the absence ofinhibitor to a concentration of 10-13 mg/ml and then subsequently addinginhibitor to the protein provided a protein sample that wouldcrystallize albeit at a slower rate.

Sparse matrix screening of pQE70-BACE in the presence of the inhibitorshown in FIG. 1 at 20° C. in the hanging drop vapor diffusion method wasperformed with the commercially available Wizard I screen (EmeraldBiostructures, Bainbridge Island, Wash.) and Hampton I screen (HamptonResearch, Laguna Nigel, Calif.). A shower of microcrystals was observedin Wizard I screen condition 45 (20% PEG 3000 (precipitant), 0.1 Msodium acetate pH 4.5 (buffer)) and Hampton I screen condition 37 (8%PEG 4000 (precipitant), 0.1 M sodium acetate pH 4.6 (buffer)). Firstround optimization experiments, pQE70-BACE produced crystals in 0.1 Msodium acetate pH 4.5-5.6 (buffer) and PEG 2000, 3000, 4000, and 8000(precipitants) at 20° C. Initial crystals grown in PEG 3000 consisted ofrod clusters and single rod shaped crystals with a large depletion. Acrystal grown in 16% PEG 3000 (precipitant) and 0.1 M sodium acetate pH4.6 (buffer) at 20° C., with an approximate size of 0.6×0.15 mm,diffracted to 1.7 Å at the Argonne National Laboratory. The cryogenicsolution for this crystal consisted of synthetic mother liquor based onthe well solution and glycerol: 16% PEG 3000, 0.1 M sodium acetate pH4.6, and 5-30% glycerol (cryoagent) in 5% increments. The cryogenicsolutions were added stepwise in 5-minute increments with increasingpercentages of glycerol. The crystal was looped out and flash frozen inliquid nitrogen. The crystal contains one molecule per asymmetric unitwith cell dimensions of a=73.1 Å, b=105.1 Å, c=50.5 Å, α=90°, β=94.8°,γ=90° in space group C2.

Second round optimization also resulted in large single crystals, withan approximate size of 0.3-0.4×0.12-0.2 mm, in 4-6% PEG 4000 and PEG8000 (precipitants), 0.1 M sodium acetate pH 4.6-5.6 (buffers) at 20° C.The typical cryogenic solution for these crystals consisted of onepercentage higher of the same PEG condition found in the well motherliquor of the crystal, 0.1 M sodium acetate (pH of the mother liquor),and 25% glycerol. The cryogenic solutions were added every 5 minuteswith stepwise additions of 0.1 μl, 0.25 μl, 0.50 μl, 1.0 μl, and 2.0 μl.After a one hour soak, crystals were looped out and flash frozen inliquid nitrogen. These crystals have the same unit cell as the pET11acrystals (a=81 Å, b=103 Å, c=100 Å, α=γ=90, β=105°) with a space groupof P2₁ and three molecules per asymmetric unit but diffracted to lowerresolution (2.5-2.8 Å) than the C2 crystal form, discussed above.

Third round optimization revealed that the percentage of PEG(precipitant) was the critical component required to distinguish betweenthe C2 and P2₁ crystal forms. The greater the percentage of precipitantpresent, the more dehydrated the crystals become resulting in higherresolution diffraction (the solvent content in the P2₁ crystals is 54%compared to the 42% solvent content for the C2 crystals).Crystallization in 8% PEG or less reproducibly gave the lower resolutionP2₁ crystal form while crystallization in 16% PEG or more (up to 45%PEG) reproducibly gave the higher resolution C2 crystal form.Alternative PEGs such as PEG 200, PEG 350 MME, PEG 400, PEG 550 MME, PEG750 MME, PEG 1000, PEG 2000, PEG 2000 MME, PEG 3000, PEG 4000, PEG 8000also produce suitable crystals when used with pQE70-BACE. The buffer forcrystallizing with the different forms of PEG was 0.1 M sodium acetatepH 4.6-5.6 at a temperature of 20° C. No additional salt was requiredfor crystallization. In addition, streak seeding at the stage of settingup the hanging drops aided crystal growth. A range of proteinconcentration from 2 mg/ml to 13 mg/ml has proven useful in preparingcrystals.

X-Ray Diffraction Characterization

Initial data collection was carried out on home source X-rays using aRigaku RUH3R X-ray generator (with osmic confocal mirros) and a R-axisIV++ detector (Molecular Structure Corporation, The Woodlands, Tex.).Initial analysis of the crystals revealed 1.9 Å diffraction on the homesource. The same crystal was refrozen and transported to the synchrotronfor subsequent data collection at the Advanced Photon Source (Argonne,Ill.) at beamline 17-ID. Using synchrotron radiation, the crystaldiffracted to 1.7 Å resolution. Crystals were of the space group C2 withcell dimensions of a=73.1 Å, b=105.1 Å, c=50.5 Å, α=90°, β=94.8°, γ=90°.The Matthews coefficient for these crystals assuming that there is onemolecule in the asymmetric unit is 2.1 Å/Da with 42% solvent. Thestructure determination (see below) revealed the presence of electrondensity in the active site appropriate for the inhibitor shown in FIG.1.

Molecular Replacement

The structure was solved by molecular replacement. A solution wasdetermined using AMORE (Navaza, Acta Cryst., D50:157-63 (1994);Collaborative Computational Project N4, Acta Cryst. D50:760-3 (1994)) byutilizing a previously solved structure of human BACE produced in E.coli from the pET11a vector. The initial rotation solution gave a singlestrong peaks of 16.9σ. A translation search in space group C2 resultedin a correlation coefficient of 57.2 with an R-factor of 38.3% to 4 Åresolution. The high correlation coefficient and low R-factor suggestedthat the entire protein contents of the unit cell had been correctlyidentified; therefore, the search for additional molecules wasabandoned.

TABLE 10 Data collection statistics for 1.9 Å resolution data set ofHuman BACE derived from E. coli pQE70-BACE (C2 crystal form) producedprotein used for refinement (data collected at λ 1.54 Å on home sourceX-rays). Data was processed with D*trek. Rmerge vs Resolution ResolutionAverage Num Num Num Num <<I>/ ChiSq Rmerge Rmerge range counts obs rejsovlps mults <sig>> norm shell cumul 19.97-4.08  33303 11727 46 116662975 10.3 0.35 0.039 0.039 4.08-3.25 27063 11593 43 11536 2913 9.6 0.480.048 0.043 3.25-2.84 11447 11354 44 11298 2839 8.1 0.75 0.066 0.0472.84-2.58 6285 11441 77 11343 2852 6.8 0.99 0.080 0.049 2.58-2.39 418811239 98 11112 2785 5.8 1.22 0.097 0.052 2.39-2.25 3250 11130 123 109752753 5.1 1.30 0.111 0.054 2.25-2.14 2298 11102 164 10893 2732 4.4 1.400.131 0.056 2.14-2.05 1621 9532 111 9355 2350 3.7 1.41 0.149 0.0572.05-1.97 1278 6190 95 6012 1527 3.6 1.26 0.147 0.058 1.97-1.90 798 413147 4004 1026 3.1 1.16 0.174 0.058 19.97-1.90  10512 99439 848 9819424752 6.5 1.00 0.058 0.058 Redundancy vs Resolution Resolution CalcPercent of reflections measured N times, N = % Comp % Comp range unique0 1 2 3 4 5-8 9-12 >12 shell cumul 19.97-4.08  3039 1.6 0.5 5.3 0.6 88.53.5 0.0 0.0 98.4 98.4 4.08-3.25 3011 2.8 0.5 3.2 0.8 89.5 3.3 0.0 0.097.2 97.8 3.25-2.84 2964 3.8 0.4 2.4 0.6 89.3 3.5 0.0 0.0 96.2 97.32.84-2.58 3013 4.6 0.7 2.5 0.5 88.4 3.3 0.0 0.0 95.4 96.8 2.58-2.39 29745.4 1.0 2.0 0.7 87.2 3.8 0.0 0.0 94.6 96.4 2.39-2.25 2963 6.0 1.1 2.10.7 86.4 3.7 0.0 0.0 94.0 96.0 2.25-2.14 2974 6.6 1.5 2.1 0.7 85.4 3.70.0 0.0 93.4 95.6 2.14-2.05 2986 19.1 2.2 1.4 1.6 72.7 2.9 0.0 0.0 80.993.8 2.05-1.97 2985 46.1 2.8 1.7 2.0 45.2 2.2 0.0 0.0 53.9 89.31.97-1.90 2978 62.9 2.7 1.2 2.2 29.9 1.2 0.0 0.0 37.1 84.1 19.97-1.90 29887 15.9 1.3 2.4 1.0 76.3 3.1 0.0 0.0 84.1 84.1 Percent of reflectionsResolution measured AT LEAST N times, N = range 13 9 5 4 3 2 119.97-1.90 0.0 0.0 3.1 79.4 80.4 82.8 84.1

TABLE 11 Data collection statistics for 1.7 Å resolution dataset ofHuman BACE derived from E. coli pQE70-BACE (C2 crystal form) producedprotein used for the intial molecular replacement solution (datacollected at λ 1.0000 Å at APS, 17-ID). Data was processed with HKL2000.Summary of reflections intensities and R-factors by shells R linear =SUM (ABS(I − <I>))/SUM (I) R square = SUM ((I − <I>) ** 2)/SUM (I ** 2)Chi**2 = SUM ((I − <I>) ** 2)/(Error ** 2 * N/(N − 1))) In all sumssingle measurements are excluded Shell limit Lower Upper Average AverageNorm. Linear Square Angstrom I error stat. Chi**2 R-fac R-fac 50.00 3.66 37330.4 701.7 364.7 2.455 0.039 0.045 3.66 2.91 18841.1 418.8 278.72.645 0.054 0.059 2.91 2.54 7573.0 231.6 188.0 2.262 0.069 0.074 2.542.31 4744.8 196.2 174.2 1.911 0.085 0.088 2.31 2.14 3766.7 199.8 185.11.655 0.097 0.099 2.14 2.02 2682.2 195.6 186.9 1.324 0.115 0.110 2.021.91 1738.0 182.7 178.3 1.079 0.143 0.131 1.91 1.83 1038.1 170.3 168.20.833 0.183 0.159 1.83 1.76 665.1 166.9 165.9 0.691 0.235 0.200 1.761.70 625.6 219.6 219.0 0.608 0.220 0.205 All reflections 8296.7 272.2211.6 1.651 0.060 0.052 Shell I/Sigma in resolution shells: Lower Upper% of reflections with I/Sigma less than limit limit 0 1 2 3 5 10 20 >20total 50.00  3.66 0.1 0.3 0.5 0.6 0.7 1.7 4.2 93.9 98.0 3.66 2.91 0.40.9 1.4 2.1 3.6 7.3 16.0 82.5 98.6 2.91 2.54 1.0 2.4 3.8 5.4 8.0 16.134.0 64.0 98.0 2.54 2.31 1.6 3.7 6.8 9.4 15.0 27.8 51.2 46.5 97.7 2.312.14 2.2 5.6 9.0 12.7 20.3 36.3 62.3 34.8 97.1 2.14 2.02 2.3 8.1 14.319.6 29.6 49.6 75.4 21.3 96.7 2.02 1.91 4.0 12.6 20.9 28.6 42.2 63.584.6 11.7 96.3 1.91 1.83 6.8 20.8 32.8 42.2 56.1 75.9 89.9 5.4 95.3 1.831.76 7.6 25.7 40.6 51.6 64.0 80.2 88.7 2.0 90.6 1.76 1.70 5.5 19.7 33.243.6 54.5 64.1 67.7 0.3 68.0 All hkl 3.1 10.0 16.3 21.5 29.3 42.2 57.336.4 93.7Model Building and Refinement

Further rigid body refinement of the model in CNX (MolecularSimulations, Inc) followed by minimization refinement gave an R-factorof 37.5% and a Free R-factor of 40.1% to 4.0 Å with an overall B-factorof 25.0 Å². Minimization and B-factor refinement led to a R-factor of30.7% and a Free R-factor of 33.4%. The subsequent availability of ahigher resolution data set to 1.7 Å afforded the opportunity to continuethe high resolution refinement of the structure. The refinement againsthigher resolution data was initiated with a rigid body refinementfollowed by minimization and B-factor refinement leading to a R-factorof 29.6% and a Free R-factor of 30.7%. Including a round of simulatedannealing refinement (A. T. Brunger, A. Krukowski, J. W. Erickson ActaCryst A 46:585-93, (1990)) and minimization led to an improved R-factorof 26.3% and a Free R-factor of 28.3%. During each cycle of refinement abulk solvent correction was incorporated (J. S. Jiang & A. T. Brunger,J. Mol. Biol. 243:100-15 (1994)). Progress of the refinement wasmonitored by a decrease in both the R-factor and Free R-factor.

At this point, inspection of the electron density map within the activesite revealed electron density that was unaccounted for by the proteinmodel and consistent with the shape of the inhibitor shown in FIG. 1that was present in the crystallization conditions. Model building wasdone using the program CHAIN (J. S. Sack, Journal of Molecular Graphics6:224-5 (1988)) and LORE (B. C. Finzel, Meth. Enzymol. 277:230-42(1997)). Rebuilding of the model and the addition of water moleculesinto the model using the 1.7 Å resolution map afforded the opportunityfor further cycles of refinement (including the inhibitor) givingimprovement of the R-factor to 20.6% and a Free R-factor of 23.1%. Themodel includes three residues from the N terminal pro-region (61P-63P),residues 1-157, 165-309, 317-386. Loops for residues 158-164 and 310-316were disordered in the electron density and therefore have been omittedfrom the model.

TABLE 12 Refinement Statistics for structure of Human BACE from pQE70(C2 crystal form). R-factor Free R-factor No. of reflections 20-1.70 Å F≧ 2σ 0.206 0.231 37857 Bonds (Å) Angles (°) r.m.s deviation from idealgeometry 0.006 1.4 Number of atoms Average B-factor Protein 2982 24.6Waters 370 35.5 Ligand 41 18.7 Total 3393 25.8

Example 3 Crystallization and Structure Determination of Human BACE inP4₃2₁2 crystal form

Examples of the crystallization and structure determination of humanbeta secretase in the P43212 crystal form are disclosed in U.S.Provisional Application Ser. No. 60/290,107, filed May 10, 2001 and U.S.patent application Ser. No. 10/143,502, filed on the same day herewith,and entitled “CRYSTALLIZATION AND STRUCTURE DETERMINATION OF BETASECRETASE AND/OR BETA SECRETASE-LIKE PROTEINS”.

Expression, Purification, and Crystallization

Production of Recombinant Human γ-Secretase in CHO-K1 Cells. The codingsequence was engineered to delete the terminal transmembrane andcytoplasmic domain and introduce a C-terminal hexahistidine tag usingthe polymerase chain reaction. The 5′ sense oligonucleotide primer[CGCTTTGGATCCGTGGACAACCTGAGGGGCAA] (SEQ ID NO:6) was designed toincorporate a BamHI site for ease in subcloning and Kozak consensussequence around the initiator methionine for optimal translationinitiation. The 3′ antisense primer[CGCTTTGGTACCCTATGACTCATCTGTCTGTGGAATGTTG] (SEQ ID NO:7) incorporated ahexahistidine tag and translation termination codon just upstream of thepredicted transmembrane domain (Ser⁴³²) and a NotI restriction site forcloning. The PCR was performed on the plasmid templatepcDNA3.1hygroAsp2R for 15 cycles [94° C., 30 sec., 65° C., 30 sec., 72°C., 30 sec] using Pwo I polymerase (Roche Biochemicals, Indianapolis,Ind.) as outlined by the manufacturer. The PCR product was digested tocompletion with BamHI and NotI and ligated into the BamHI and NotI sitesof the Baculovirus transfer vector pVL1393 (PharMingen, San Diego,Calif.). A portion of the ligation was used to transform competent E.coli DH5α cells and recombinant clones were selected on ampicillin.Individual clones containing the proper cDNA inserts were identified byPCR. Plasmid DNA from clone (pVL1393/Hu_Asp-2LΔTM(His)₆) was prepared byalkaline lysis and banding in CsCl. The integrity of the insert wasconfirmed by complete DNA sequencing. For CHO-K1 cell expression,plasmid pVL1393/Hu_Asp-2LΔTM(His)₆ was digested with BamHI and NotI andthe resulting fragment subcloned into the mammalian expression vectorpcDNA3.1(hygro) as described above to yieldpcDNA3.1(hygro)/Hu_Asp-2LΔTM(His)₆).

For expression, CHO-K1 cells (50% confluent) were transfected withcationic liposome/pcDNA3.1(hygro)/Hu_Asp-2LΔTM(His)₆ complexes in α-MEMmedium containing 10% FBS overnight. Selection was performed in the samemedium containing 0.5 mg/L hygromycin B for seven days and survivingcells were cloned by limiting dilution. Eight cell lines were screenedfor soluble β-secretase by Western blot analysis using a polyclonalrabbit antiserum specific for human β-secretase (UP-191). Conditionedmedium from each clonal cell line was concentrated by Ni⁺-NTA (resinavailable under the trade designation FAST FLOW from Qiagen, Valencia,Calif.) chromatography and the histidine-tagged polypeptide eluted withbuffer containing 50 mM imidazole. Aliquots of the latter fraction weredisplayed on a PVDF membrane and recombinant soluble human β-secretasewas visualized using UP-191 antiserum and alkaline phosphataseconjugated goat antirabbit second antibody. Based on these results,clone #4 showed the highest expression level and was used for allsubsequent experiments.

Purification of Recombinant Human/Secretase from CHO-K1 Cells. Forpurification, the medium was concentrated approximately 10-fold using atangential flow concentrator equip with a 30,000 molecular weight cutoffcartridge. Solid ammonium sulfate was then slowly added with stirring tothe concentrate at 4° C. to a final value of 40% saturation (242 g/L).After stirring at 4° C. for 30 minutes, the suspension was clarified bycentrifugation (16,000×g, 60 minutes) and the supernatant taken forfurther analysis. The 40% ammonium sulfate supernatant was adjusted to80% saturation by slow addition of solid ammonium sulfate with stirringat 4° C. (281 g/L). After stirring for 30 minutes at 4° C., theinsoluble material was collected by centrifugation as indicated aboveand the 40-80% ammonium sulfate pellet taken for further analysis.

The 40-80% ammonium sulfate pellet was dissolved in 25 mM Tris-HCl (pH8.5)/0.5 M NaCl/10 mM imidazole ( 1/10 the original volume) and appliedto a 12.5 ml column containing Ni⁺-NTA resin (available under the tradedesignation FAST FLOW from Qiagen, Valencia, Calif.) previouslyequilibrated in the same buffer. Following sample application, thecolumn was washed with 10 column volumes of loading buffer and theneluted with 25 mM Tris-HCl (pH 8.5)/0.5 M NaCl/50 mM imidazole. Thematerial eluting in 50 mM imidazole was pooled, concentratedapproximately 10-fold using a YM 30 membrane (30,000 MWCO), and thendialyzed against 10 mM HEPES-Na (pH 8.0) using 50,000 molecular weightcutoff tubing. For affinity purification, the synthetic peptideSer-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Arg-Gly-Gly-Cys (where Sta—statine)(FIG. 2, SEQ ID NO:4) was synthesized and coupled to SULFO-LINK® resin(Pierce Chemical Company, Rockford, Ill.) as recommended by themanufacture. The dialyzed material from above was adjusted to 0.1 MNaOAc (pH 4.5) by addition of 1/10 volume of 1.0 M NaOAc (pH 4.5) andimmediately applied to the synthetic peptide shown in FIG. 2/SULFO-LINK®column (6 ml containing 1.0 mg of the synthetic peptide shown in FIG.2/ml of resin) that had been previously equilibrated in 25 mM NaOAc (pH4.5). Following sample application, the column was washed with 10 columnvolumes of 25 mM NaOAc (pH 4.5) and then eluted with 50 mM sodium borate(pH 8.5). N-terminal sequence analysis of the affinity purified materialrevealed an equimolar mixture of pro- and processed human β-secretasebeginning at Thr¹ and Glu²⁵, respectively. The final proteinconcentration was determined by amino acid analysis assuming a 52 kDaglycoprotein for insect cells and a 60 kDa glycoprotein for CHO cells,respectively.

Cleavage of Beta Secretase by HIV Protease. HIV-1 protease is able tocleave beta secretase at F³⁹—V⁴⁰ bond as shown in FIG. 11. It wasreasoned that a homogeneous preparation of beta secretase with V⁴⁰ as anN-terminus could be made if conditions could be found to bring thecleavage to completion. The optimal conditions for cleavage weredetermined by examining the N-terminal sequence of beta secretase afterincubation with HIV-1 protease in various conditions. These included apH range of 4-7, urea concentrations of 0.5-3.0 M, and time incrementsof 15 minutes, up to 2 hours incubation at 37° C. It was found that atpH 5.7, 0.5 M urea, with 5% HIV protease and 1 hour incubation at 37°C., nearly 100% cleavage of the F³⁹—V⁴⁰ bond occurred. The processedenzyme was separated from the other components of the reaction mixtureby using an affinity column based on the synthetic peptide shown in FIG.2 and dialysis. Beta secretase produced by the technique described aboveis about 10-20% more active than unprocessed beta secretase.

In a typical experiment, 25 mg (3.85×10⁻⁷ moles) of CHO cell derivedbeta secretase in 4.65 ml (at 5.38 mg/ml per amino acid analysis) wastreated with a 10% molar equivalent of the HIV-1 protease dimer(3.85×10⁻⁸ moles, 7.69×10⁻⁴ g) in the presence of 0.5 M urea and 0.2 MMES (pH 5.7). Before adding the HIV-1 protease, the beta secretase wasprepared in the following manner: 1) 4.65 ml of beta secretase was addedto 1.163 ml of 1M MES (pH 5.7) with no precipitate being observed; 2)0.55 ml of 6 M urea was added to the mixture in step 1 while stirringwith some precipitate being observed; 3) 0.222 ml of HIV-1 protease(×3.47 mg/ml=7.70×10⁻⁴ g). The mixture was then incubated for 2 hours at37° C.

Further purification after HIV protease treatment. Afterwards, themixture was dialyzed overnight versus 0.5 M urea and 0.2 M MES (pH 5.7)at 4° C. using a membrane available under the trade designationSPECTRA/POR 6® Membrane, MWCO: 50,000 (Part No. 132-544) from SpectrumLaboratories (Rancho Dominguez, Calif.). The next morning, the samplewas changed into a solution containing 10 mM MES (pH 5.7) and 50 mM NaCl(no urea) at 4° C., and allowed to continue dialyzing for an additional8 hours. At this point, the solution was spun to remove the precipitate,and the supernatant analyzed by absorbance at 280 nm. It was estimated,using a conversion factor of 0.685 mg/mg AU, that approximately 20.4 mgof material was present to carry forward to the next, and final stage ofpurification. 20.4 mg of beta secretase in 9.3 ml (containing 10 mM MES(pH 5.7) and 50 mM NaCl (no urea) at 4° C.) from the preceding stepswere added to 2.325 ml of 1 M sodium acetate (pH 4.5). It was thenapplied to a 2 ml affinity column that had been pre-equilibrated with0.2 M sodium acetate (pH 4.5). The affinity column was made by couplingthe synthetic peptide shown in FIG. 2 (1 mg/ml of resin) to 2 ml of aresin available under the trade designation SULFO-LINK® from PierceChemical Co. ((Rockford, Ill.). The flow through material wasrecirculated 2× before washing the column with 20 mM solution (pH 4.5)and 150 mM NaCl. The beta secretase was eluted into 6 ml of 0.1 M sodiumborate (pH 8.5). Absorbance at 280 nm indicated a total of 13.6 mg ofbeta secretase. The affinity column was re-equilibrated, and the processrepeated using the flow through. Another 6 ml containing a total of 5.1mg was recovered (according to absorbance at 280 nm). Thus a combinedtotal of 18.7 mg was realized after treatment of the CHO cell derivedbeta secretase with HIV-1 protease, and subsequent purification. Thus, acombined total of 18.7 mg was realized after treatment of the CHO cellderived beta secretase with HIV-1 protease, and subsequent purification.

Purified HIV-1 protease treated (and non-treated) beta secretase wereassayed for activity b HPLC analysis of the products. Purified HIV-1protease treated beta secretase showed 10-20% more enzymatic activitythan non-treated beta secretase.

Crystallization. The crystallization conditions for HIV protease treatedCHO produced beta secretase were found by trying to reproduce untreatedCHO cell beta secretase crystallization conditions. The untreated formof the protein contains a 50:50 mixture of pro and processed forms. Atray was set up to attempt to reproduce the crystallization conditionsalong with test crystallization conditions that were cryogenic. The trayhad a row that was 20-25% PEG 2000 MME, 100 mM sodium acetate (pH 4.5),a 0.75 microliter+0.75 microliter reservoir+protein drop, with a 500microliter reservoir volume in a crystallization plate and an incubationtemperature of 20° C. The other three rows were the same as the firstwith the addition of 10% DMSO for the second row, 10% ethylene glycolfor the third row, and 10% glycerol for the fourth row. The tray wasstreak seeded by using a cat whisker and a seed stock from untreated CHOcrystals. Within a week crystals were observed in the second, third, andfourth rows. After three weeks, small crystals were observed in thefirst row. Later experiments demonstrated that seeding was notnecessary. The crystals observed without seeding were of the same formas crystals formed from seeding. Crystals obtained from seedingexperiments were of a different crystal form than the original untreatedCHO crystals used for seeds.

Detailed Crystallization Method. The protein was obtained in 20 mM HEPES(pH 8.0) and 50 mM NaCl at approximately 1 mg/ml for crystallizationstudies. During concentration to approximately 25 mg/ml the protein wasexchanged into 20 mM HEPES (NaOH, pH 7.75) and the NaCl concentrationwas diluted to less than 1 mM.

Crystals were grown from the following conditions: protein+compoundpreparation−20 mg/ml protein, 2 mM of the inhibitor shown in FIG. 1(dissolved in 100% DMSO), 10% DMSO (including DMSO from compound); wellsolution: 17-28% PEG 2000 MME or PEG 5000 MME, 50-200 mM sodium acetate(pH 4.5), 0-20% glycerol or 10% ethylene glycol or 10% DMSO or 10% MPD.

Crystallization method: 500 microliters reservoir volume in a hangingdrop vapor diffusion tray (plates available under the trade designationVDX plate and CRYSCHEM® plate from Hampton Research, Laguna Niguel,Calif.). The protein and reservoir solutions were added together in a1:1 ratio on the coverslip or sitting drop post. Drop size of 0.75microliter+0.75 microliter was preferred, although 0.75-1.5 microliterswell+1.5-2 microliters protein was also sufficient to produce crystals.The trays were then stored in a 20° C. incubator for the nucleation andgrowth phases. Crystals appeared in 7-10 days (with the inhibitor shownin FIG. 1) with final size of 0.3 mm×0.3mm occurring by day 21.

Cryo conditions: With optimal crystallization conditions, the drop wascryogenic (did not form crystalline ice). Reservoir: 20% PEG 2000 MME,100 mM sodium acetate (pH 4.5), and 10% glycerol. Protein preparation:20 mg/ml protein, 2 mM of the inhibitor shown in FIG. 1, and 10% DMSO.Crystals that were frozen directly from the crystallization drop did notdiffract as well as a crystal mounted in a capillary tube. Thediffraction limit of the frozen crystal was 6 Å while a crystal ofsimilar size (0.2 mm×0.2 mm) diffracted to better than 4 Å when mountedin a capillary tube. Optimization of cryo conditions included addingsupplemental amounts of a cryo agent to the crystallization drop. Thepreferred cryo agent was ethylene glycol, however glycerol or DMSO alsoprovided cryoprotection. The method for adding supplemental cryo agentto the drop was as follows: 1 microliter 100% cryo agent was added to 3microliters of reservoir solution to give a 25% cryo stock; the mixturewas allowed to set for 1 minute before 0.5 microliter of the mix wasadded to a 0.75 microliter+0.75 microliter crystallization drop; afterwaiting an additional 3 minutes, another 1 microliter of the mixture wasadded. The second addition of the cryo mix brought the cryoconcentration to 12.5%. Cryopreservation was completed by looping outthe crystal after about 3 minutes to 2 hours and freezing the crystal byplunging the loop into liquid nitrogen.

X-Ray Diffraction Characterization

All data collection was carried out at the Advanced Photon Source(Argonne, Ill.) at beamline 17-ID. The crystals diffracted to 2.9 Åusing synchrotron radiation. Crystals were of the space group P4₃2₁2with cell constants a=114.0±20 Å, b=114±20 Å, c=190±20 Å, and α=γ=90°.The Matthews coefficient for these crystals assuming that there are twomolecules in the asymmetric unit is 2.4 Å/Da with 48% solvent. Thestructure determination (see below) revealed the presence of electrondensity in the active site appropriate for the inhibitor shown in FIG.1.

Molecular Replacement

A molecular replacement solution was determined using AMORE (Navaza,Acta Cryst., D50:157-63 (1994); Collaborative Computational Project N4,Acta Cryst. D50:760-63 (1994)) by utilizing a previously solvedstructure of human beta secretase from CHO crystals. The initialrotation solution gave a single strong peak of 7.8σ with the nextstrongest peak appearing at 6.8σ and the third strongest peak appearingat 4.0σ. The presence of two strong peaks suggested that two moleculesmight be present in the asymmetric unit. The final determination of thespace group (P4₁2₁2 or P4₃2₁2) was determined experimentally by testingtranslation searches in each space group. A translation search in thecorrect space group, P4₃2₁2, resulted in a correlation coefficient of41.7 with an R-factor of 46.3% to 4 Å resolution for the first molecule.A translation search for the second molecule resulted in an improvedcorrelation coefficient of 60.9 with an R-factor of 38.0 to 4 Åresolution for both molecules.

TABLE 13 Data collection statistics for structure of Human BetaSecretase (data collected at λ 1.0000 Å at APS, 17-ID) Shell limit LowerUpper Average Average Norm. Linear Square Angstrom I error stat. Chi**2R-fac R-fac 20.00  5.98 6592.1 110.7 86.2 2.032 0.044 0.047 5.98 4.773833.6 60.8 49.6 2.020 0.060 0.065 4.77 4.17 3918.4 70.5 60.1 1.9330.067 0.074 4.17 3.79 2396.8 57.0 51.5 1.377 0.078 0.079 3.79 3.521453.6 52.5 49.9 0.869 0.094 0.094 3.52 3.32 853.1 51.3 50.1 0.505 0.1210.114 3.32 3.15 477.4 49.6 49.2 0.320 0.169 0.160 3.15 3.02 296.8 48.948.7 0.232 0.234 0.215 3.02 2.90 178.4 52.9 52.8 0.198 0.358 0.343 2.902.80 153.4 86.7 86.7 0.277 0.442 0.474 All reflections 2130.2 63.7 57.71.089 0.071 0.062Model Building and Refinement

Further rigid body refinement of the model in CNX (MolecularSimulations, Inc) followed by minimization and group b-factor refinementgave an R-factor of 30.9% and a Free R-factor of 35.6% to 2.9 Å. Duringeach cycle of refinement a bulk solvent correction was incorporated(Jiang et al., J. Mol. Biol. 243:100-15 (1994)). Progress of therefinement was monitored by a decrease in both the R-factor and FreeR-factor.

At this point, inspection of the electron density map within the activesite revealed electron density that was unaccounted for by the proteinmodel and consistent with the shape of the inhibitor shown in FIG. 1that was present in the crystallization conditions. Model building wasdone using the program CHAIN (Sack, Journal of Molecular Graphics,6:224-25 (1988)) and LORE (Finzel, Meth. Enzymol., 277:230-42 (1997)).Modest rebuilding of the model into the 2.9 Å resolution map affordedthe opportunity for further cycles of refinement (including theinhibitor) giving marginal improvement of the R-factor to 30.7% and aFree R-factor of 36.0%. Residues 158-170 and 311 to 316 were disorderedin the electron density and therefore have been omitted from the model.

TABLE 14 Refinement Statistics for structure of Human Beta SecretaseR-factor Free R-factor No. of reflections 20-2.9 Å F ≧ 2σ 0.307 0.360027670 Bonds (Å) Angles (°) r.m.s deviation from ideal 0.012 1.8 geometryAverage B- Number of atoms factor Protein 5768 74.3 Ligand 82 18.7 Total5850 73.5

The complete disclosure of all patents, patent applications includingprovisional applications, and publications, and electronically availablematerial (e.g., GenBank amino acid and nucleotide sequence submissions;and protein data bank (pdb) submissions) cited herein are incorporatedby reference. The foregoing detailed description and examples have beengiven for clarity of understanding only. No unnecessary limitations areto be understood therefrom. The invention is not limited to the exactdetails shown and described; many variations will be apparent to oneskilled in the art and are intended to be included within the inventiondefined by the claims.

SEQUENCE LISTING FREE TEXT SEQ ID NO:1 residues for the E. coliexpressed recombinant human BACE produced from the pET11a construct SEQID NO:2 residues for the E. coli expressed recombinant human BACEproduced from the pQE80L construct SEQ ID NO:3 residues for the E. coliexpressed recombinant human BACE produced from the pQE70 construct SEQID NO:4 synthetic peptide SEQ ID NO:5 residues for CHO cell expressedrecombinant human beta secretase proteolytically cleaved with HIVprotease (used for crystallization) SEQ ID NO:6 5′ sense oligonucleotideprimer SEQ ID NO:7 3′ antisense oligonucleotide primer

1. A method of using an isolated crystal of beta secretase (BACE) of SEQID NO:3 having space group symmetry C2, unit cell dimensions of a, b,and c, wherein a is about 53 Å to about 93 Å, b is about 85 Å to about125 Å, and c is about 40 Å to about 60 Å; α=γ=90°, β is about 85° toabout 105°; selecting a potential modifier by performing rational drugdesign with a three-dimensional structure determined for the crystal,wherein selecting is performed in conjunction with computer modeling;adding the potential modifier to a binding assay; and detecting ameasure of binding, wherein the potential modifier that binds isselected as a potential drug.
 2. A method of using an isolatedco-crystal of beta secretase (BACE) of SEQ ID NO:3 having space groupsymmetry C2, unit cell dimensions of a, b, and c, wherein a is about 53Å to about 93 Å, b is about 85 Å to about 125 Å, and c is about 40 Å toabout 60 Å; α=γ=90°, β is about 85° to about 105°; selecting a potentialmodifier by performing rational drug design with a three-dimensionalstructure determined for the crystal, wherein selecting is performed inconjunction with computer modeling; adding the potential modifier to abinding assay; and detecting a measure of binding, wherein the potentialmodifier that binds is selected as a potential drug.
 3. The method ofclaim 2 wherein the exposing comprises soaking.
 4. The method of claim 2wherein the samples include a variety of different functional groups. 5.The method of claim 3, wherein the crystal of BACE wherein a is 73.1 Å,b=105.1 Å, c=50.5 Å, α=γ=90°, and β=94.8°.
 6. The method of claim 2further comprising identifying the ligand that forms the ligand-BACEmolecular complex.
 7. The method of claim 6 wherein one of thedetermining and identifying comprises collecting x-ray diffraction data.8. The method of claim 7 wherein one of the determining and identifyingcomprises calculating an electron density function.
 9. A method of usingan isolated crystal of beta secretase (BACE) of SEQ ID NO:1 or SEQ IDNO:2 having space group symmetry P21, unit cell dimensions of a, b, andc, wherein a is about 61 Å to about 101 Å, b is about 83 Å to about 123Å, and c is about 80 Å to about 120 Å; α=γ=90°, β is about 95° to about115°; selecting a potential modifier by performing rational drug designwith a three-dimensional structure determined for the crystal, whereinselecting is performed in conjunction with computer modeling; adding thepotential modifier to a binding assay; and detecting a measure ofbinding, wherein the potential modifier that binds is selected as apotential drug.
 10. A method of using an isolated co-crystal of betasecretase (BACE) of SEQ ID NO:1 or SEQ ID NO :2 having space groupsymmetry P2₁, unit cell dimensions of a, b, and c, wherein a is about 61Å to about 101 Å, b is about 83 Å to about 123 Å, and c is about 80 Å toabout 120 A; α=γ=90°, β is about 95° to about 115°; selecting apotential modifier by performing rational drug design with athree-dimensional structure determined for the crystal, whereinselecting is performed in conjunction with computer modeling; adding thepotential modifier to a binding assay; and detecting a measure ofbinding, wherein the potential modifier that binds is selected as apotential drug.
 11. The method of claim 10 wherein the exposingcomprises soaking.
 12. The method of claim 11 wherein the samplesinclude a variety of different functional groups.
 13. The method ofclaim 10 further comprising identifying the ligand that forms theligand-BACE molecular complex.
 14. The method of claim 13 wherein one ofthe determining and identifying comprises collecting x-ray diffractiondata.
 15. The method of claim 13 wherein one of the determining andidentifying comprises calculating an electron density function.